Lettuce named carrizo

ABSTRACT

Novel lettuce, such as lettuce designated CARRIZO is disclosed. In some embodiments, the disclosure relates to the seeds of lettuce designated CARRIZO, to the plants and plant parts of lettuce designated CARRIZO, and to methods for producing a lettuce plant by crossing the lettuce designated CARRIZO with itself or another lettuce plant. The disclosure further relates to methods for producing a lettuce plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other lettuce plants derived from the lettuce designated CARRIZO.

FIELD

The present disclosure relates to the field of agriculture, to new anddistinctive lettuce (Lactuca sativa) cultivars, such as a cultivardesignated CARRIZO, and to methods of making and using such plants.

BACKGROUND

Lettuce is an important and valuable vegetable crop. Thus, a continuinggoal of plant breeders is to develop stable, high yielding lettucecultivars that are agronomically sound or unique. The reasons for thisgoal are to maximize the amount of yield produced on the land used aswell as to improve the plant agronomic and horticultural qualities. Toaccomplish this goal, the lettuce breeder must select and developlettuce plants that have the traits that result in superior cultivars.

SUMMARY OF THE DISCLOSURE

The following embodiments and aspects thereof are described inconjunction with systems, tools and methods which are meant to beexemplary, not limiting in scope.

According to the disclosure, in some embodiments, there is provided anovel lettuce cultivar, designated CARRIZO, also interchangeablyreferred to as ‘lettuce plant CARRIZO,’ ‘lettuce CARRIZO’, ‘lettucenamed CARRIZO,’ or ‘CARRIZO’.

This disclosure thus relates to the seeds of lettuce cultivar designatedCARRIZO, to the plants or parts of lettuce cultivar designated CARRIZO,to the plants or parts thereof comprising all the physiological andmorphological characteristics of lettuce cultivar designated CARRIZO orparts thereof. The plants or parts of lettuce cultivar designatedCARRIZO have all the physiological and morphological characteristics oflettuce cultivar designated CARRIZO and/or have one or morecharacteristics of lettuce cultivar designated CARRIZO including but notlimited to as determined at the 5% significance level when grown in thesame environmental conditions, and/or have one or more physiological andmorphological characteristics of lettuce cultivar designated CARRIZOincluding but not limited to as determined at the 5% significance levelwhen grown in the same environmental conditions, and/or have all thephysiological and morphological characteristics of lettuce cultivardesignated CARRIZO when grown in the same environmental conditionsand/or have one or more physiological and morphological characteristicsof lettuce cultivar designated CARRIZO when grown in the sameenvironmental conditions and/or have all of the physiological andmorphological characteristics of lettuce cultivar designated CARRIZOwhen grown in the same environmental conditions. The disclosure alsorelates to variants, mutants and trivial modifications of lettucecultivar designated CARRIZO and parts thereof. In some embodiments, arepresentative sample of seed of lettuce designated CARRIZO is depositedunder NCIMB No.______

Plant parts of the lettuce cultivar designated CARRIZO of the presentdisclosure are also provided, such as, but not limited to, a head, leaf,a flower, a seed, a cell, pollen or an ovule obtained from the plantcultivar. The present disclosure provides heads and/or leaves of thelettuce cultivar designated CARRIZO. Such heads and/or leaves or partsthereof could be used as fresh products for consumption or in processesresulting in processed products such as food products comprising one ormore harvested parts of the lettuce plant CARRIZO, for example harvestedleaves and/or heads. The harvested parts or food products can be or cancomprise the lettuce heads and/or leaves of the lettuce plant CARRIZO ora salad mixture comprising leaves of the lettuce plant CARRIZO. The foodproducts might have undergone one or more processing steps such as, butnot limited to cutting, washing, mixing, etc. All such products are partof the present disclosure. The present disclosure also provides plantparts or cells of the lettuce cultivar designated CARRIZO, wherein aplant regenerated from said plants parts or cells has one or more, orall the phenotypic and morphological characteristics of the lettuceplant CARRIZO, such as one or more, or all the characteristics of thelettuce plant CARRIZO including but not limited to as determined at the5% significance level when grown in the same environmental conditions.All such parts and cells are part of the present disclosure.

The plants and seeds of the present disclosure include those that may beof an essentially derived variety as defined in section 41(3) of thePlant Variety Protection Act of The United States of America, e.g., avariety that is predominantly derived from lettuce cultivar designatedCARRIZO or from a variety that i) is predominantly derived from lettucecultivar designated CARRIZO, while retaining the expression of theessential characteristics that result from the genotype or combinationof genotypes of lettuce cultivar designated CARRIZO; ii) is clearlydistinguishable from lettuce cultivar designated CARRIZO; and iii)except for differences that result from the act of derivation, conformsto the initial variety in the expression of the essentialcharacteristics that result from the genotype or combination ofgenotypes of the lettuce cultivar designated CARRIZO.

In another aspect, the present disclosure provides regenerable cells. Insome embodiments, the regenerable cells are for use in tissue culture oflettuce cultivar designated CARRIZO. In some embodiments, the tissueculture is capable of regenerating plants comprising all thephysiological and morphological characteristics of lettuce cultivardesignated CARRIZO, and/or having all the physiological andmorphological characteristics of lettuce cultivar designated CARRIZO,and/or having one or more physiological and morphologicalcharacteristics of lettuce cultivar designated CARRIZO, and/or havingall of the physiological and morphological characteristics of lettucecultivar designated CARRIZO. In some embodiments, the regenerated plantshave the characteristics of lettuce cultivar designated CARRIZOincluding but not limited to as determined at the 5% significance levelwhen grown in the same environmental conditions and/or have all thephysiological and morphological characteristics of lettuce cultivardesignated CARRIZO including but not limited to as determined at the 5%significance level when grown in the same environmental conditionsand/or have one or more physiological and morphological characteristicsof lettuce cultivar designated CARRIZO including but not limited to asdetermined at the 5% significance level when grown in the sameenvironmental conditions and/or have all of the physiological andmorphological characteristics of lettuce cultivar designated CARRIZO butnot limited to as determined at the 5% significance level when grown inthe same environmental conditions. In some embodiments, the plant partsand cells used to produce such tissue cultures will be embryos,meristematic cells, seeds, callus, pollens, leaves, anthers, pistils,roots, root tips, stems, petioles, heads, cotyledons, hypocotyls,ovaries, seed coats, fruits, endosperms, flowers, axillary buds or thelike. Protoplasts produced from such tissue culture are also included inthe present disclosure. The lettuce heads, leaves, shoots, roots andwhole plants regenerated from the tissue culture, as well as the headsand leaves produced by said regenerated plants are also part of thedisclosure. In some embodiments, the whole plants regenerated from thetissue culture have one, more than one, or all the physiological andmorphological characteristics of lettuce cultivar designated CARRIZO,including but not limited to as determined at the 5% significance levelwhen grown in the same environmental conditions.

The disclosure also discloses methods for vegetatively propagating aplant of the present disclosure. In the present application,vegetatively propagating can be interchangeably used with vegetativereproduction. In some embodiments, the methods comprise collecting partsof a lettuce cultivar designated CARRIZO and regenerating a plant fromsaid parts. In some embodiments, the part can be for example a leaf. Insome embodiments, the methods can be for example cutting that is rootedinto an appropriate medium according to techniques known by the oneskilled in the art. Plants and plant parts thereof, including but notlimited to leaves and heads thereof, produced by such methods are alsoincluded in the present disclosure. In another aspect, the plants andparts thereof such as leaves and heads thereof produced by such methodscomprise all the physiological and morphological characteristics oflettuce cultivar designated CARRIZO, and/or have all of thephysiological and morphological characteristics of lettuce cultivardesignated CARRIZO, and/or have the physiological and morphologicalcharacteristics of lettuce cultivar designated CARRIZO, and/or have oneor more of the characteristics of lettuce cultivar designated CARRIZO.In some embodiments, plants produced by such methods consist of one,more than one, or all the physiological and morphologicalcharacteristics of lettuce cultivar designated CARRIZO, including butnot limited to as determined at the 5% significance level when grown inthe same environmental conditions.

Further included in the disclosure are methods for producing heads,leaves and/ or seeds from the lettuce cultivar designated CARRIZO. Insome embodiments, the methods comprise growing a lettuce cultivardesignated CARRIZO to produce lettuce heads, leaves and/or seeds. Insome embodiments, the methods further comprise harvesting the lettuceheads, and/or seeds. Such lettuce heads, leaves and/or seeds are part ofthe present disclosure. In some embodiments, the lettuce heads, leavesand/or seeds have all the physiological and morphologicalcharacteristics of the lettuce heads, leaves and/or seeds of lettucecultivar designated CARRIZO (e.g. those listed in Table 1 and/ordeposited under NCIBM No.______) when grown in the same environmentalconditions and/or have one or more physiological and morphologicalcharacteristics of the lettuce head, leaves and/or seeds of lettucecultivar designated CARRIZO (e.g. those listed in Table 1 and/ordeposited under NCIBM No.______) when grown in the same environmentalconditions and/or have the characteristics of the lettuce head, leaves,and/or seeds of lettuce cultivar designated CARRIZO (e.g. those listedin Table 1 and/or deposited under NCIBM No.______) when grown in thesame environmental conditions.

Also included in this disclosure are methods for producing a lettuceplant. In some embodiments, the lettuce plant is produced by crossingthe lettuce cultivar designated CARRIZO with itself or another lettuceplant. In some embodiments, another plant can be an inbred lettucecultivar/line or a hybrid. When crossed with itself, i.e. when CARRIZOis crossed with another lettuce cultivar CARRIZO, respectively orself-pollinated, lettuce cultivar CARRIZO will be conserved (e.g. as aninbred). When crossed with another different lettuce plant, an F1 hybridseed is produced if the different lettuce plant is an inbred and a“three-way cross” seed is produced if the different lettuce plant is ahybrid. Such F1 hybrid seed and three-way hybrid seeds and plantsproduced by growing said F1 and three-way hybrid seeds are included inthe present disclosure. Methods for producing F1 and three-way hybridlettuce seeds comprising (a) crossing lettuce cultivar CARRIZO lettuceplant with a different lettuce cultivar/line or a hybrid and (b)harvesting the resultant hybrid lettuce seed are also part of thedisclosure. The hybrid lettuce seeds produced by the methods comprisingcrossing lettuce cultivar CARRIZO lettuce plant with a different lettuceplant and harvesting the resultant hybrid lettuce seed are included inthe disclosure, as are included the hybrid lettuce plants or partsthereof and seeds produced by said grown hybrid lettuce plants.

Further included in the disclosure are methods for producing lettuceseeds and plants made thereof. In some embodiments, the methods compriseself-pollinating the lettuce cultivar CARRIZO and harvesting theresultant seeds. Lettuce seeds produced by such method are also part ofthe disclosure.

In another embodiment, this disclosure also relates to methods forproducing other lettuce plants derived from lettuce cultivar CARRIZO andto the lettuce plants derived by the use of those methods.

In some embodiments, such methods for producing a lettuce plant derivedfrom the lettuce cultivar CARRIZO comprise (a) self-pollinating thelettuce cultivar CARRIZO plant at least once to produce a progeny plantderived from lettuce cultivar CARRIZO. In some embodiments, the methodsfurther comprise (b) crossing the progeny plant derived from lettucecultivar CARRIZO with itself or a second lettuce plant to produce a seedof a progeny plant of a subsequent generation. In some embodiments, themethods further comprise (c) growing the progeny plant of the subsequentgeneration. In some embodiments, the methods further comprise (d)crossing the progeny plant of the subsequent generation with itself or asecond lettuce plant to produce a lettuce plant further derived from thelettuce cultivar CARRIZO. In further embodiments, step (b), step (c)and/or step (d) are repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, ormore generations to produce a lettuce plant derived from the lettucecultivar CARRIZO. In some embodiments, within each crossing cycle, thesecond plant is the same plant as the second plant in the last crossingcycle. In some embodiments, within each crossing cycle, the second plantis different from the second plant in the last crossing cycle, which canbe a lettuce variety/cultivar/line other than CARRIZO, a lettuce hybridor a plant of Lactuca genus.

Another method for producing a lettuce plant derived from the varietyCARRIZO, comprises (a) crossing the CARRIZO plant with a second lettuceplant to produce a progeny plant derived from lettuce cultivar CARRIZO.In some embodiments, the method further comprises (b) crossing theprogeny plant derived from lettuce cultivar CARRIZO with itself or asecond lettuce plant to produce a seed of a progeny plant of asubsequent generation; In some embodiments, the method further comprises(c) growing the progeny plant of the subsequent generation; In someembodiments, the method further comprises (d) crossing the progeny plantof the subsequent generation with itself or a second lettuce plant toproduce a lettuce plant derived from CARRIZO. In a further embodiment,step (b), step (c) and/or step (d) are repeated for at least 1, 2, 3, 4,5, 6, 7, 8, or more generation to produce a lettuce plant derived fromCARRIZO. In some embodiments, within each crossing cycle, the secondplant is the same plant as the second plant in the last crossing cycle.In some embodiments, within each crossing cycle, the second plant isdifferent from the second plant in the last crossing cycle, which can bea lettuce variety/cultivar/line other than CARRIZO, a lettuce hybrid ora plant of Lactuca genus.

In one aspect, the present disclosure provides methods of introducingone or more desired traits into the lettuce cultivar CARRIZO and plantsor seeds obtained from such methods. In another aspect, the presentdisclosure provides methods of introducing a single locus conversionconferring one or more desired traits into the lettuce cultivar CARRIZO,and plants or parts including heads, leaves and/or seeds obtained fromsuch methods. In further aspect, the present disclosure provides methodsof modifying a single locus and introducing one or more desired traitsinto the lettuce cultivar CARRIZO, and plants or parts including heads,leaves and/or seeds, obtained from such methods. The desired trait(s)may be, but not exclusively, conferred by a single locus that contains asingle and/or multiple gene(s). In some embodiments, the gene is adominant allele. In some embodiments, the gene is a partially dominantallele. In some embodiments, the gene is a recessive allele. In someembodiments, the gene or genes will confer such traits, including butnot limited to male sterility, herbicide resistance, insect resistance,resistance for bacterial, fungal, mycoplasma or viral disease, enhancedplant quality such as improved drought or salt tolerance, water-stresstolerance, heat-stress tolerance, improved standability, enhanced plantvigor, improved shelf life, delayed senescence or controlled ripening,enhanced nutritional quality such as increased sugar content orincreased sweetness, increased texture, improved flavor and aroma,improved fruit length and/or size, protection for color, fruit shape,uniformity, length or diameter, refinement or depth, improved yield andrecovery, improved fresh cut application, specific aromatic compounds,specific volatiles, leaf texture, and specific nutritional components.For the present disclosure and the skilled artisan, disease isunderstood to include, but not limited to fungal diseases, viraldiseases, bacterial diseases, mycoplasma diseases, or other plantpathogenic diseases and a disease resistant plant will encompass a plantresistant to fungal, viral, bacterial, mycoplasma, and other plantpathogens. In one aspect, the gene or genes may be naturally occurringgene mutation(s) and/or spontaneous or induced mutations(s). In anotheraspect, genes are mutated, modified, genetically engineered through theuse of New Breeding Techniques described herein. In some embodiments,the method for introducing the desired trait(s) is a backcrossingprocess making use of a series of backcrosses to lettuce cultivardesignated CARRIZO (a.k.a. lettuce cultivar CARRIZO or lettuce CARRIZO),which the desired trait(s) is maintained by selection. The single geneconverted plants or single locus converted plants that can be obtainedby the methods are included in the present disclosure. The single geneconversion plants that can be obtained by the methods are included inthe present disclosure.

When dealing with a gene that has been modified, for example through NewBreeding Techniques, the trait (genetic modification) could be directlymodified into the newly developed line/cultivar such as lettuce cultivarCARRIZO. Alternatively, if the trait is not modified into each newlydeveloped line/cultivar such as lettuce cultivar CARRIZO, anothertypical method used by breeders of ordinary skill in the art toincorporate the modified gene is to take a line already carrying themodified gene and to use such line as a donor line to transfer themodified gene into the newly developed line. The same would apply for anaturally occurring trait or one arising from spontaneous or inducedmutations.

In some embodiments, the backcross breeding process of lettuce cultivarCARRIZO comprises (a) crossing lettuce cultivar CARRIZO with anotherplants that comprise the desired trait(s) to produce F1 progeny plants.In some embodiments, the process further comprises (b) selecting the F1progeny plants that have the desired trait(s). In some embodiments, theprocess further comprises (c) crossing the selected F1 progeny plantswith the lettuce cultivar CARRIZO plants to produce backcross progenyplants. In some embodiments, the process further comprises (d) selectingfor backcross progeny plants that have the desired trait(s) andessentially all the physiological and morphological characteristics ofthe lettuce cultivar CARRIZO to produce selected backcross progenyplants; In some embodiments, the process further comprises (e) repeatingsteps (c) and (d) one, two, three, four, five six, seven, eight, nine ormore times in succession to produce selected, second, third, fourth,fifth, sixth, seventh, eighth, ninth or higher backcross progeny plantsthat have the desired trait(s) and essentially all the characteristicsof the lettuce cultivar CARRIZO, and/or have the desired trait(s) andessentially all the physiological and morphological characteristics ofthe lettuce cultivar CARRIZO, and/or have the desired trait(s) andessentially all the physiological and morphological characteristics ofthe lettuce cultivar CARRIZO, including but not limited to when grown inthe same environmental conditions or including but not limited to at a5% significance level when grown in the same environmental conditions.The lettuce plants or seed produced by the methods are also part of thedisclosure. Backcrossing breeding methods, well known to one skilled inthe art of plant breeding will be further developed in subsequent partsof the specification.

In an embodiment of this disclosure is a method of making a backcrossconversion of lettuce cultivar CARRIZO. In some embodiments, the methodcomprises crossing lettuce cultivar CARRIZO with a donor plantcomprising an induced gene mutation(s), a naturally occurring genemutation(s), or a gene(s) and/or sequences modified through New BreedingTechniques conferring one or more desired traits to produce F1 progenyplants. In some embodiments, the method further comprises selecting theF1 progeny plant comprising the naturally occurring gene mutation(s),the induced gene mutation(s) or gene(s) and/or sequence(s) modifiedthrough New Breeding Techniques conferring the one or more desiredtraits. In some embodiments, the method further comprises backcrossingthe selected progeny plant to the lettuce cultivar CARRIZO. This methodmay further comprise the step of obtaining a molecular marker profile ofthe lettuce cultivar CARRIZO and using the molecular marker profile toselect for the progeny plant with the desired trait and the molecularmarker profile of the lettuce cultivar CARRIZO. The plants or partsthereof produced by such methods are also part of the presentdisclosure.

In some embodiments of the disclosure, the number of loci that may betransferred and/or backcrossed into the lettuce cultivar CARRIZO is atleast 1, 2, 3, 4, 5 or more. A single locus may contain several genes. Asingle locus conversion also allows for making one or more site specificchanges to the plant genome, such as, without limitation, one or morenucleotide changes, deletions, insertions, substitutions etc. In someembodiments, the single locus conversion is performed by genome editing,a.k.a. genome editing with engineered nucleases (GEEN). In someembodiments, the genome editing comprises using one or more engineerednucleases. In some embodiments, the engineered nucleases include, butare not limited to Zinc finger nucleases (ZFNs), TranscriptionActivator-Like Effector Nucleases (TALENs), the CRISPR/Cas system (usingsuch as Cas9, Cas12a/Cpf1, Cas13/C2c2, CasX and CasY), engineeredmeganuclease, engineered homing endonucleases, and endonucleases for DNAguided genome editing (Gao et al., Nature Biotechnology (2016), doi:10.1038/nbt.3547). In some embodiments, the single locus conversionchanges one or several nucleotides of the plant genome. Such genomeediting techniques are some of the techniques now known by the personskilled in the art and herein are collectively referred to as “NewBreeding Techniques”. In some embodiments, one or more above-mentionedgenome editing methods are directly applied on a plant of the presentdisclosure, Accordingly, a cell containing an edited genome, or a plantpart containing such cell can be isolated and used to regenerate a novelplant which has a new trait conferred by said genome editing, andessentially all the physiological and morphological characteristics oflettuce cultivar CARRIZO.

The disclosure further provides methods for developing lettuce plants ina lettuce plant breeding program using plant breeding techniquesincluding but not limited to, recurrent selection, backcrossing,pedigree breeding, genomic selection, molecular marker (IsozymeElectrophoresis, Restriction Fragment Length Polymorphisms (RFLPs),Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily PrimedPolymerase Chain Reactions (AP-PCRs), DNA Amplification Fingerprintings(DAFs), Sequence Characterized Amplified Regions (SCARs), AmplifiedFragment Length Polymorphisms (AFLPs), and Simple Sequence Repeats(SSRs) which are also referred to as Microsatellites, Single NucleotidePolymorphisms (SNPs), enhanced selection, genetic markers, enhancedselection and transformation. Seeds, lettuce plants, and parts thereofproduced by such breeding methods are also part of the disclosure.

The disclosure also relates to variants, mutants and trivialmodifications of the seed or plant of the lettuce cultivar CARRIZO.Variants, mutants and trivial modifications of the seed or plant oflettuce cultivar CARRIZO can be generated by methods available to oneskilled in the art, including but not limited to, mutagenesis (e.g.,chemical mutagenesis, radiation mutagenesis, transposon mutagenesis,insertional mutagenesis, signature tagged mutagenesis, site-directedmutagenesis, and natural mutagenesis), knock-outs/knock-ins, antisenseoligonucleotides, RNA interference and other techniques such as the NewBreeding Techniques described herein. For more information ofmutagenesis in plants, such as agents or protocols, see Acquaah et al.(Principles of plant genetics and breeding, Wiley-Blackwell, 2007, ISBN1405136464, 9781405136464, which is herein incorporated by reference inits entity).

The disclosure also relates to a mutagenized population of the lettucecultivar CARRIZO and methods of using such populations. In someembodiments, the mutagenized population can be used in screening for newlettuce plants which comprise essentially one or more or all of themorphological and physiological characteristics of lettuce cultivarCARRIZO. In some embodiments, the new lettuce plants obtained from thescreening process comprise essentially all the morphological andphysiological characteristics of the lettuce cultivar CARRIZO, and oneor more additional or different morphological and physiologicalcharacteristics that lettuce cultivar CARRIZO does not have.

This disclosure is also directed to methods for producing a lettuceplant by crossing a first parent lettuce plant with a second parentlettuce plant, wherein either the first or second parent lettuce plantis a lettuce cultivar CARRIZO. Further, both first and second parentlettuce plants can come from the lettuce cultivar CARRIZO. Further, thelettuce cultivar CARRIZO can be self-pollinated i.e. the pollen of alettuce cultivar CARRIZO can pollinate the ovule of the same lettucecultivar CARRIZO, respectively. When crossed with another lettuce plant,a hybrid seed is produced. Such methods of hybridization andself-pollination are well known to those skilled in the art of breeding.

A lettuce cultivar such as lettuce cultivar CARRIZO has been producedthrough several cycles of self-pollination and is therefore to beconsidered as a homozygous plant or line. An inbred line can also beproduced though the dihaploid system which involves doubling thechromosomes from a haploid plant or embryo thus resulting in an inbredline that is genetically stable (homozygous) and can be reproducedwithout altering the inbred line: Haploid plants could be obtained fromhaploid embryos that might be produced from microspores, pollen, anthercultures or ovary cultures or spontaneous haploidy. The haploid embryosmay then be doubled by chemical treatments such as by colchicine or bedoubled autonomously. The haploid embryos may also be grown into haploidplants and treated to induce the chromosome doubling. In either case,fertile homozygous plants may be obtained. A hybrid variety isclassically created through the fertilization of an ovule from an inbredparental line by the pollen of another, different inbred parental line.Due to the homozygous state of the inbred line, the produced gametescarry a copy of each parental chromosome. As both the ovule and thepollen bring a copy of the arrangement and organization of the genespresent in the parental lines, the genome of each parental line ispresent in the resulting F1 hybrid, theoretically in the arrangement andorganization created by the plant breeder in the original parental line.

As long as the homozygosity of the parental lines is maintained, theresulting hybrid cross shall be stable. The F1 hybrid is then acombination of phenotypic characteristics issued from two arrangementand organization of genes, both created by a person skilled in the artthrough the breeding process.

Still further, this disclosure is also directed to methods for producinga lettuce cultivar CARRIZO-derived lettuce plant by crossing lettucecultivar CARRIZO with a second lettuce plant. In some embodiments, themethods further comprise obtaining a progeny seed from the cross. Insome embodiments, the methods further comprise growing the progeny seed,and possibly repeating the crossing and growing steps with the lettucecultivar CARRIZO-derived plant from 0 to 7, or more times. Thus, anysuch methods using the lettuce cultivar CARRIZO are part of thisdisclosure: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using lettuce cultivarCARRIZO as a parent are within the scope of this disclosure, includingplants derived from lettuce cultivar CARRIZO. In some embodiments, suchplants have one, more than one, or all of the physiological andmorphological characteristics of lettuce cultivar designated CARRIZOincluding but not limited to as determined at the 5% significance levelwhen grown in the same environmental conditions. In some embodiments,such plants might exhibit additional and desired characteristics ortraits such as high seed yield, high seed germination, seedling vigor,early maturity, high yield, disease tolerance or resistance, andadaptability for soil and climate conditions. Consumer-driven traits,such as a preference for a given head and or leaf size, shape, color,texture, taste, are other traits that may be incorporated into newlettuce plants developed by this disclosure.

A lettuce plant can also be propagated vegetatively. A part of theplant, for example a shoot or a leaf tissue, is collected, and a newplant is obtained from the part. Such part typically comprises an apicalmeristem of the plant. The collected part is transferred to a mediumallowing development of a plantlet, including for example rooting ordevelopment of shoots. This is achieved using methods well-known in theart. Accordingly, in one embodiment, a method of vegetativelypropagating a plant of the present disclosure comprises collecting apart of a plant according to the present disclosure, e.g. a shoottissue, and obtaining a plantlet from said part. In one embodiment, amethod of vegetatively propagating a plant of the present disclosurecomprises: (a) collecting tissue of a plant of the present disclosure;(b) rooting said proliferated shoots to obtain rooted plantlets. In oneembodiment, a method of vegetatively propagating a plant of the presentdisclosure comprises: (a) collecting tissue of a plant of the presentdisclosure; (b) cultivating said tissue to obtain proliferated shoots;(c) rooting said proliferated shoots to obtain rooted plantlets. In oneembodiment, such method further comprises growing a plant from saidplantlets. In one embodiment, a head is harvested from said plant. Inone embodiment, a leaf is harvested from said plant. In one embodiment,a seed is harvested from said plant. In one embodiments, such plants,heads, leaves and/or seeds have all the physiological and morphologicalcharacteristics of plants, heads, leaves and/or seeds of lettucecultivar CARRIZO when grown in the same environmental conditions. In oneembodiment, the head is processed into products with prepared cut headsand leaves.

In some embodiments, the present disclosure teaches a seed of lettucecultivar CARRIZO, wherein a representative sample of seed of saidlettuce cultivar is deposited under NCIMB No.______

In some embodiments, the present disclosure teaches a lettuce plant, ora part thereof, produced by growing the deposited CARRIZO seed.

In some embodiments, the present disclosure teaches a lettuce plantpart, wherein the lettuce part is selected from the group consisting of:a leaf, a flower, a head, a seed, an ovule, a pollen, and a cell.

In some embodiments, the present disclosure teaches a lettuce plant, ora part thereof, having all the characteristics of lettuce cultivarCARRIZO deposited under NCIMB No.______ of this disclosure including butnot limited to as determined at the 5% significance level when grown inthe same environmental conditions.

In some embodiments, the present disclosure teaches a lettuce plant, ora part thereof, having all the physiological and morphologicalcharacteristics of lettuce cultivar CARRIZO, wherein a representativesample of seed of said lettuce plant was deposited under NCIMB No.______

In some embodiments, the present disclosure teaches a tissue culture ofregenerable cells produced from the plant or plant part grown from thedeposited lettuce cultivar CARRIZO seed, wherein cells of the tissueculture are produced from a plant part selected from the groupconsisting of protoplasts, embryos, meristematic cells, callus, pollens,ovules, flowers, seeds, leaves, roots, root tips, anthers, stems,petioles, heads, axillary buds, cotyledons and hypocotyls. In someembodiments, the plant part includes protoplasts produced from a plantgrown from the deposited lettuce cultivar CARRIZO seed.

In some embodiments, the present disclosure teaches a compositioncomprising regenerable cells produced from the plant or part thereofgrown from the deposited lettuce cultivar CARRIZO seed, or other plantpart or plant cell thereof. In some embodiments, the compositioncomprises a growth media. In some embodiments, the growth media is solidor a synthetic cultivation medium. In some embodiments, the compositionis a lettuce plant regenerated from the tissue culture from a plantgrown from the deposited lettuce cultivar CARRIZO seed, said planthaving all of the characteristics of lettuce cultivar CARRIZO, wherein arepresentative sample of seed of said lettuce cultivar CARRIZO isdeposited under NCIMB No.______

In some embodiments, the present disclosure teaches lettuce headsproduced from plants grown from the deposited lettuce cultivar CARRIZOseeds.

In some embodiments, the present disclosure teaches lettuce leavesproduced from plants grown from the deposited lettuce cultivar CARRIZOseeds.

In some embodiments, the methods of producing said lettuce head comprise(a) growing the lettuce plant from deposited lettuce cultivar CARRIZOseed to produce a lettuce head, and (b) harvesting said lettuce head. Insome embodiments, the present disclosure also teaches a lettuce headproduced by the method of producing lettuce head as described above. Insome embodiments, such heads have all the physiological andmorphological characteristics of heads of lettuce cultivar CARRIZO (e.g.those listed in Table 1 and/or deposited under NCIMB No.______) whengrown in the same environmental conditions.

In some embodiments, the methods of producing said lettuce leaf comprise(a) growing the lettuce plant from deposited lettuce cultivar CARRIZOseed to produce a lettuce leaf, and (b) harvesting said lettuce leaf. Insome embodiments, the present disclosure also teaches a lettuce leafproduced by the method of producing lettuce leaf as described above. Inone embodiment, such leaves have all the physiological and morphologicalcharacteristics of leaves of lettuce cultivar CARRIZO (e.g. those listedin Table 1 and/or deposited under NCIMB No.______) when grown in thesame environmental conditions.

In some embodiments, the present disclosure teaches methods forproducing a lettuce seed comprising crossing a first parent lettuceplant with a second parent lettuce plant and harvesting the resultantlettuce seed, wherein said first parent lettuce plant and/or secondparent lettuce plant is the lettuce plant produced from the depositedlettuce cultivar CARRIZO seed, or a lettuce plant having all thecharacteristics of lettuce cultivar CARRIZO (listed in Table 1 and/ordeposited under NCIMB No._______) including but not limited to asdetermined at the 5% significance level when grown in the sameenvironmental conditions.

In some embodiments, the present disclosure teaches methods forproducing a lettuce seed comprising self-pollinating the lettuce plantgrown from the deposited lettuce cultivar CARRIZO seed and harvestingthe resultant lettuce seed.

In some embodiments, the present disclosure teaches the seed produced byany of the above described methods.

In some embodiments, the present disclosure teaches methods ofvegetatively propagating the lettuce plant grown from the depositedlettuce cultivar CARRIZO seed, said method comprising (a) collecting apart of a plant grown from the deposited lettuce cultivar CARRIZO seedand (b) regenerating a plant from said part.

In some embodiments, the method further comprises harvesting a head fromsaid vegetatively propagated plant. In some embodiments, the methodfurther comprises harvesting a leaf from said vegetatively propagatedplant.

In some embodiments, the present disclosure teaches the plant, the headsand leaves of plants vegetatively propagated from plant parts of plantsgrown from the deposited lettuce cultivar CARRIZO seed. In someembodiments, such plant, heads and/or leaves have all the physiologicaland morphological characteristics of lettuce cultivar CARRIZO plant,head and/or leaves of lettuce cultivar CARRIZO (e.g. those listed inTable 1 and/or deposited under NCIMB No.______) when grown in the sameenvironmental conditions.

In some embodiments, the present disclosure teaches methods of producinga lettuce plant derived from the lettuce cultivar CARRIZO. In someembodiment the methods comprise (a) self-pollinating the plant grownfrom the deposited lettuce cultivar CARRIZO seed at least once toproduce a progeny plant derived from lettuce cultivar CARRIZO. In someembodiments, the method further comprises (b) crossing the progeny plantderived from lettuce cultivar CARRIZO with itself or a second lettuceplant to produce a seed of a progeny plant of a subsequent generationand; (c) growing the progeny plant of the subsequent generation from theseed, and (d) crossing the progeny plant of the subsequent generationwith itself or a second lettuce plant to produce a lettuce plant derivedfrom the lettuce cultivar CARRIZO. In some embodiments said methodsfurther comprise the step of: (e) repeating steps (c) and/or (d) for atleast 1, 2, 3, 4, 5, 6, 7 or more generation to produce a lettuce plantderived from the lettuce cultivar CARRIZO.

In some embodiments, the present disclosure teaches methods of producinga lettuce plant derived from the lettuce cultivar CARRIZO, the methodscomprising (a) crossing the plant grown from the deposited lettucecultivar CARRIZO seed with a second lettuce plant to produce a progenyplant derived from the lettuce cultivar CARRIZO. In some embodiments,the method further comprises (b) crossing the progeny plant derived fromthe lettuce cultivar CARRIZO with itself or a second lettuce plant toproduce a seed of a progeny plant of a subsequent generation and; (c)growing the progeny plant of the subsequent generation from the seed;(d) crossing the progeny plant of the subsequent generation with itselfor a second lettuce plant to produce a lettuce plant derived from thelettuce cultivar CARRIZO. In some embodiments said methods furthercomprise the steps of: (e) repeating steps (b), (c) and/or (d) for atleast 1, 2, 3, 4, 5, 6, 7 or more generations to produce a lettuce plantderived from the lettuce cultivar CARRIZO.

In some embodiments, the present disclosure teaches plants grown fromthe deposited lettuce cultivar CARRIZO seed wherein said plants comprisea single locus conversion. As used herein, the term “a” or “an” refersto one or more of that entity; for example, “a single locus conversion”refers to one or more single locus conversions or at least one singlelocus conversion. As such, the terms “a” (or “an”), “one or more” and“at least one” are used interchangeably herein. In addition, referenceto “an element” by the indefinite article “a” or “an” does not excludethe possibility that more than one of the elements are present, unlessthe context clearly requires that there is one and only one of theelements.

In some embodiments, the present disclosure teaches a method ofproducing a plant of lettuce plant designated CARRIZO comprising atleast one desired trait, the method comprising introducing a singlelocus conversion conferring the desired trait into lettuce plantdesignated CARRIZO, whereby a plant of lettuce designated CARRIZOcomprising the desired trait is produced.

In some embodiments, the present disclosure teaches a lettuce plant,comprising a single locus conversion and essentially all of thecharacteristics of lettuce plant designated CARRIZO deposited underNCIMB No.______. In other embodiments, the single locus conversion isintroduced into the plant by the use of recurrent selection, mutationbreeding, wherein said mutation breeding selects for a mutation that isspontaneous or artificially induced, backcrossing, pedigree breeding,haploid/double haploid production, marker-assisted selection, genetictransformation, genomic selection, synthetic genomics, Zinc fingernuclease (ZFN) technology, oligonucleotide directed mutagenesis,cisgenesis, intragenesis, RNA-dependent DNA methylation,agro-infiltration, Transcription Activation-Like Effector Nuclease(TALEN), CRISPR/Cas system, engineered meganuclease, engineered homingendonuclease, and/or DNA guided genome editing.

In some embodiments, the plant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,,or more single locus conversions. In some embodiments, the plantcomprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 single locusconversions, but essentially all of the other physiological andmorphological characteristics of lettuce plant CARRIZO and/or depositedunder NCIMB No.______. In some embodiments, the plant comprises at leastone single locus conversion and essentially all of the physiological andmorphological characteristics of lettuce plant CARRIZO deposited underNCIMB No.______. In other embodiments, the plant comprises one singlelocus conversion and essentially all of the other physiological andmorphological characteristics of lettuce plant CARRIZO deposited underNCIMB No.______

In some embodiments said single locus conversion confers said plantswith a trait selected from the group consisting of male sterility, malefertility, herbicide resistance, insect resistance, resistance forbacterial, fungal, mycoplasma or viral disease, enhanced plant qualitysuch as improved drought or salt tolerance, water stress tolerance, heattolerance, improved standability, enhanced plant vigor, improved shelflife, delayed senescence or controlled ripening, increased nutritionalquality such as increased sugar content or increased sweetness,increased texture, improved flavor and aroma, improved fruit lengthand/or size, protection for color, fruit shape, uniformity, length ordiameter, refinement or depth lodging resistance, improved yield andrecovery when compared to a suitable check/comparison plant. In someembodiments, the check plant is a lettuce cultivar CARRIZO not havingsaid single locus conversion. In some embodiments, the at least onesingle locus conversion is a naturally-occurring spontaneous mutation,an artificially induced mutation or a gene or nucleotide sequencemodified through the use of New Breeding Techniques.

In some embodiments, the present disclosure teaches methods forproducing nucleic acids, comprising isolating nucleic acids from theplant grown from the deposited CARRIZO seed, or a part, or a cellthereof. In some embodiments, the present disclosure teaches methods forproducing a second lettuce plant, comprising applying plant breedingtechniques to the plant grown from the deposited lettuce seed, or partthereof to produce the second lettuce plant.

In some embodiments, the present disclosure provides a method ofproducing a commodity plant product comprising collecting the commodityplant product from the plant of the present disclosure. The commodityplant product produced by said method is also part of the presentdisclosure.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

DETAILED DESCRIPTION OF THE DISCLOSURE

The following description includes information that may be useful inunderstanding the present disclosure. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed disclosures, or that any publication specifically orimplicitly referenced is prior art.

Definitions

In the description and tables that follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Adaptability: A plant that has adaptability is a plant able to grow wellin different growing conditions (climate, soils, etc.).

Allele: An allele is any of one or more alternative forms of a genewhich relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing: Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotypes of the F₁hybrid.

Big Vein: Big vein is a disease of lettuce caused by Mirafiori lettuceBig Vein Virus (MiLBVV, genus Ophiovirus) which is transmitted by thefungus Olpidium virulentus, with vein clearing and leaf shrinkageresulting in plants of poor quality and reduced marketable value.

Bolting: The premature development of a flowering stalk, and subsequentseed, before a plant produces a food crop. Bolting is typically causedby late planting when temperatures are low enough to cause vernalizationof the plants

Collection of seeds: In the context of the present disclosure acollection of seeds is a grouping of seeds mainly containing similarkind of seeds, for example inbred line seeds of the disclosure, but thatmay also contain, mixed together with another different kind of seeds,for example parental line seeds or hybrid seeds.

Commodity plant product: A “commodity plant product” refers to anycomposition or product that is comprised of material derived from aplant, seed, plant cell, or plant part of the present disclosure.Commodity plant products may be sold to consumers and can be viable ornonviable. Nonviable commodity products include but are not limited tononviable seeds and grains; processed seeds, seed parts, and plantparts; dehydrated plant tissue, frozen plant tissue, and processed planttissue; seeds and plant parts processed for animal feed for terrestrialand/or aquatic animal consumption, oil, meal, flour, flakes, bran,fiber, paper, tea, coffee, silage, crushed of whole grain, and any otherfood for human or animal consumption; biomasses and fuel products; andraw material in industry.

Core diameter: Core diameter is the diameter of the internal leaf stemmeasured at the base of the head.

Core length: The core length is the length of the internal lettuce stemmeasured from the base of the cut and trimmed head to the tip of thestem.

Corky root: Corky root is a disease caused by the bacterium Sphingomonassuberifaciens, which causes the entire taproot to become brown, severelycracked, and non-functional.

Cupping: In romaine lettuce, cupping is the process by which the romaineforms a heart. Leaves of similar size are formed in the center of thehead and then, the tops of the leaves fold downwards slightly to form aheart.

Decreased vigor: A plant having a decreased vigor in the presentdisclosure is a plant that, compared to other plants has a less vigorousappearance for vegetative and/or reproductive characteristics includingshorter plant height, small fruit size, fewer fruit or othercharacteristics.

Earliness: The earliness relates the number of days from seeding toharvest.

Essentially all of the physiological and morphological characteristics:A plant having essentially all of the physiological and morphologicalcharacteristics means a plant having all of the physiological andmorphological characteristics of a plant of the present disclosure,except for example, additional traits and/or mutations which do notmaterially affect the plant of the present disclosure, or, a desiredcharacteristic(s), which can be indirectly obtained from another plantpossessing at least one single locus conversion via a conventionalbreeding program (such as backcross breeding) or directly obtained byintroduction of at least one single locus conversion via New BreedingTechniques. In some embodiments, one of the non-limiting examples for aplant having (and/or comprising) essentially all of the physiologicaland morphological characteristics shall be a plant having all of thephysiological and morphological characteristics of a plant of thepresent disclosure other than desired, additionaltrait(s)/characteristic(s) conferred by a single locus conversionincluding, but not limited to, a converted or modified gene.

First water date: The date the seed first receives adequate moisture togerminate. This can and often does equal the planting date.

Frame diameter: For frame diameter in case of romaine lettuce, themeasurement is taken from the outer most leaf tip horizontally to theouter most leaf tip. In case of icebergs, frame diameter is measuredwith the outer leaves intact.

Head diameter: Diameter of the cut and trimmed head, sliced vertically,and measured at the widest point perpendicular to the stem in case ofromaine lettuce. In case of icebergs, head diameter is measured afterremoving the outer leaves (just the round head).

Head height: Height of the cut and trimmed head, sliced vertically, andmeasured from the base of the cut stem to the cap leaf.

Head weight: Weight of saleable lettuce head, cut and trimmed to marketspecifications.

Immunity to disease(s) and or insect(s) : A lettuce plant which is notsubject to attack or infection by specific disease(s) and or insect(s)is considered immune.

Intermediate resistance to disease(s) and or insect(s) : A lettuce plantthat restricts the growth and development of specific disease(s) and orinsect(s), but may exhibit a greater range of symptoms or damagecompared to resistant plants. Intermediate resistant plants will usuallyshow less severe symptoms or damage than susceptible plant varietieswhen grown under similar environmental conditions and/or specificdisease(s) and or insect(s) pressure, but may have heavy damage underheavy pressure. Intermediate resistant lettuce plants are not immune tothe disease(s) and or insect(s).

Lettuce Mosaic virus: A disease that can cause a stunted, deformed, ormottled pattern in young lettuce and yellow, twisted, and deformedleaves in older lettuce.

Lettuce Yield (Tons/Acre) : The yield in tons/acre is the actual yieldof the lettuce at harvest.

Maturity (Date) : Maturity refers to the stage when plants are of fullsize or optimum weight, and in marketable form or shape to be ofcommercial or economic value. In leaf types they range from 50-75 daysfrom time of seeding, depending upon the season of the year. In othertypes, they range from 65-105 days from time of seeding, depending uponthe season of the year

Nasonovia ribisnigri: A lettuce aphid that colonizes the innermostleaves of the lettuce plant, contaminating areas that cannot be treatedeasily with insecticides. Two biotypes of lettuce aphid have been knownin Europe since 2007 and were designated by The Netherlands InspectionService for Horticulture (Naktuinbow) as Nr:0 and Nr:1.

New Breeding Techniques: New breeding techniques (NBTs) are said ofvarious new technologies developed and/or used to create newcharacteristics in plants through genetic variation, the aim beingtargeted mutagenesis, that is targeted introduction of new genes or genesilencing. The following breeding techniques are within the scope ofNBTs: targeted sequence changes facilitated through the use of Zincfinger nuclease (ZFN) technology (ZFN-1, ZFN-2 and ZFN-3, see U.S. Pat.No. 9,145,565, incorporated by reference in its entirety),Oligonucleotide directed mutagenesis (ODM, a.k.a., site-directedmutagenesis), Cisgenesis and intragenesis, epigenetic approaches such asRNA-dependent DNA methylation (RdDM, which does not necessarily changenucleotide sequence but can change the biological activity of thesequence), Grafting (on GM rootstock), Reverse breeding,Agro-infiltration for transient gene expression (agro-infiltration“sensu stricto”, agro-inoculation, floral dip), genome editing withendonucleases such as chemical nucleases, meganucleases, ZFNs, andTranscription Activator-Like Effector Nucleases (TALENs, see U.S. Pat.Nos. 8,586,363 and 9,181,535, incorporated by reference in theirentireties), the CRISPR/Cas system (using such as Cas9, Cas12a/Cpf1,Cas13/C2c2, CasX and CasY; also see U.S. Pat. Nos. 8,697,359; 8,771,945;8,795,965; 8,865,406; 8,871,445; 8,889,356; 8,895,308; 8,906,616;8,932,814; 8,945,839; 8,993,233; and 8,999,641, which are all herebyincorporated by reference), engineered meganuclease, engineered homingendonucleases, DNA guided genome editing (Gao et al., NatureBiotechnology (2016), doi: 10.1038/nbt.3547, incorporated by referencein its entirety), and Synthetic genomics. A major part of today’stargeted genome editing, another designation for New BreedingTechniques, is the applications to induce a DNA double strand break(DSB) at a selected location in the genome where the modification isintended. Directed repair of the DSB allows for targeted genome editing.Such applications can be utilized to generate mutations (e.g., targetedmutations or precise native gene editing) as well as precise insertionof genes (e.g., cisgenes, intragenes, or transgenes). The applicationsleading to mutations are often identified as site-directed nuclease(SDN) technology, such as SDN1, SDN2 and SDN3. For SDN1, the outcome isa targeted, non-specific genetic deletion mutation: the position of theDNA DSB is precisely selected, but the DNA repair by the host cell israndom and results in small nucleotide deletions, additions orsubstitutions. For SDN2, a SDN is used to generate a targeted DSB and aDNA repair template (a short DNA sequence identical to the targeted DSBDNA sequence except for one or a few nucleotide changes) is used torepair the DSB: this results in a targeted and predetermined pointmutation in the desired gene of interest. As to the SDN3, the SDN isused along with a DNA repair template that contains new DNA sequence(e.g. gene). The outcome of the technology would be the integration ofthat DNA sequence into the plant genome. The most likely applicationillustrating the use of SDN3 would be the insertion of cisgenic,intragenic, or transgenic expression cassettes at a selected genomelocation. A complete description of each of these techniques can befound in the report made by the Joint Research Center (JRC) Institutefor Prospective Technological Studies of the European Commission in 2011and titled “New plant breeding techniques - State-of-the-art andprospects for commercial development”, which is incorporated byreference in its entirety.

Plant adaptability: A plant having good plant adaptability means a plantthat will perform well in different growing conditions and seasons.

Plant cell: As used herein, the term “plant cell” includes plant cellswhether isolated, in tissue culture or incorporated in a plant or plantpart.

Plant part: As used herein, the term “plant part”, “part thereof” or“parts thereof” includes plant cells, plant protoplasts, plant celltissue cultures from which lettuce plants can be regenerated, plantcalli, plant clumps and plant cells that are intact in plants or partsof plants, such as embryos, pollens, ovules, flowers, seeds, heads,rootstocks, scions, stems, roots, anthers, pistils, root tips, leaves,meristematic cells, axillary buds, hypocotyls, cotyledons, ovaries, seedcoats, endosperms and the like. In some embodiments, the plant part atleast comprises at least one cell of said plant. In some embodiments,the plant part is further defined as a pollen, a meristem, a cell or anovule. In some embodiments, a plant regenerated from the plant part hasall of the phenotypic and morphological characteristics of a lettuce ofthe present disclosure, including but not limited to as determined atthe 5% significance level when grown in the same environmentalconditions.

Quantitative Trait Loci (QTL): Quantitative trait loci refer to geneticloci that control to some degree numerically representable traits thatare usually continuously distributed.

Ratio of head height/diameter: The ratio is the head height divided bythe head diameter and is an indication of the head shape; <1 isflattened, 1=round, and >1 is pointed.

Regeneration: Regeneration refers to the development of a plant fromtissue culture.

Resistance to disease(s) and or insect(s): A lettuce plant thatrestricts the growth and development of specific disease(s) and orinsect(s) under normal disease(s) and or insect(s) attack pressure whencompared to susceptible plants. These lettuce plants can exhibit somesymptoms or damage under heavy disease(s) and or insect(s) pressure.Resistant lettuce plants are not immune to the disease(s) and orinsect(s).

RHS: RHS refers to the Royal Horticultural Society of England whichpublishes an official botanical color chart quantitatively identifyingcolors according to a defined numbering system. The chart may bepurchased from Royal Hort. Society Enterprise Ltd. RHS Garden; Wisley,Woking, Surrey GU236QB, UK.

Single locus converted (conversion): Single locus converted (conversion)plants refer to plants which are developed by a plant breeding techniquecalled backcrossing, wherein essentially all of the desiredmorphological and physiological characteristics of a plant are recoveredin addition to a single locus transferred into the plant via thebackcrossing technique or via genetic engineering. A single locusconverted plant can also be referred to a plant with a single locusconversion obtained though simultaneous and/or artificially inducedmutagenesis or through the use of New Breeding Techniques described inthe present disclosure. In some embodiments, the single locus convertedplant has essentially all of the desired morphological and physiologicalcharacteristics of the original variety in addition to a single locusconverted by spontaneous and/or artificially induced mutations, which isintroduced and/or transferred into the plant by the plant breedingtechniques such as backcrossing. In other embodiments, the single locusconverted plant has essentially all of the desired morphological andphysiological characteristics of the original variety in addition to asingle locus, gene or nucleotide sequence(s) converted, mutated,modified or engineered through the New Breeding Techniques taughtherein. In the present disclosure, single locus converted (conversion)can be interchangeably referred to single gene converted (conversion).

Susceptible to disease(s) and or insect(s): A lettuce plant that issusceptible to disease(s) and or insect(s) is defined as a lettuce plantthat has the inability to restrict the growth and development ofspecific disease(s) and or insect(s). Plants that are susceptible willshow damage when infected and are more likely to have heavy damage undermoderate levels of specific disease(s) and or insect(s).

Tip burn: Means a browning of the edges or tips of lettuce leaves thatis a physiological response to a lack of calcium.

Tolerance to abiotic stresses: A lettuce plant that is tolerant toabiotic stresses has the ability to endure abiotic stress withoutserious consequences for growth, appearance and yield.

Uniformity: Uniformity, as used herein, describes the similarity betweenplants or plant characteristics which can be a described by qualitativeor quantitative measurements.

Variety: A plant variety as used by one skilled in the art of plantbreeding means a plant grouping within a single botanical taxon of thelowest known rank which can be defined by the expression of thecharacteristics resulting from a given genotype or combination ofphenotypes, distinguished from any other plant grouping by theexpression of at least one of the said characteristics and considered asa unit with regard to its suitability for being propagated unchanged(International convention for the protection of new varieties of plants). The term “variety” can be interchangeably used with “cultivar” or“line”.

Lettuce Plants

Most cultivated forms of lettuce belong to the highly polymorphicspecies Lactuca sativa which is grown for its edible head and leaves. Asa crop, lettuce is grown commercially wherever environmental conditionspermit the production of an economically viable yield. Lettuce is theworld’s most popular salad. In the United States, the principal growingregions are California and Arizona which produce approximately 329,000acres out of a total annual acreage of more than 333,000 acres (USDA,2005). Fresh lettuce is available in the United States year-roundalthough the greatest supply is from May through October. For plantingpurposes, the lettuce season is typically divided into three categories,early, mid and late, with the coastal areas planting from January toAugust, and the desert regions from August to December. Lettuce isconsumed nearly exclusively as fresh, raw product, and occasionally as acooked vegetable. Baby leaf or spring mix lettuce is an increasinglypopular crop as worldwide baby leaf lettuce consumption continues toincrease. Spring mix lettuce refers to lettuce that is grown in highconcentrations and harvested at a very young or ‘baby leaf stage,typically 30 to 45 days after planting. The plantings are often done onwider 80 to 84 inch beds and often contain up to one million plants peracre. Compared to iceberg or romaine plantings, where they are typicallyharvested 60 to 100 days after planting, with a population of roughly25,000 to 30,000 plants per acre. Spring mix plantings often includemultiple types of lettuces, all harvested when the leaves are young andtender. These plantings can include green romaine, red romaine, darklolla rossa, tango, green leaf, and red leaf types. Spring mix fieldsare most often harvested mechanically and the harvested leaves arepacked in plastic totes, where they are transported to a processingfacility where they are washed, processed and mixed according to thesalad recipe.

Lactuca sativa is in the Cichorieae tribe of the Asteraceae (Compositaefamily). Lettuce is related to chicory, sunflower, aster, dandelion,artichoke, and chrysanthemum. Sativa is one of about 300 species in thegenus Lactuca. There are several morphological types of lettuce. TheCrisphead group includes the Iceberg and Batavian types. Iceberg lettucehas a large, firm head with a crisp texture and a white or creamy yellowinterior. Batavian lettuce predates Iceberg lettuce and has a smallerand less firm head. The Butterhead group has a small, soft head with analmost oily texture. Romaine lettuce, also known as Cos lettuce, haselongated upright leaves forming a loose, loaf-shaped head and the outerleaves are usually dark green. Leaf lettuce comes in many varieties,none of which form a head. There are three types of lettuce which areseldom seen in the United States: Latin lettuce, which looks like across between Romaine and Butterhead; Stem lettuce, which has long,narrow leaves and thick, edible stems; and Oilseed lettuce, which is aprimitive type of lettuce grown for its large seeds that are pressed toobtain oil.

Lactuca sativa is normally a simple diploid species with nine pairs ofchromosomes (2N=18). However, haploidy and polyploidy lettuce plants arealso part of the present disclosure. Lettuce is an obligateself-pollinating species which means that pollen is shed before stigmaemergence, assuring 100% self-fertilization. Since each lettuce floweris an aggregate of about 10-20 individual florets (typical of theCompositae family), manual removal of the anther tubes containing thepollen is tedious. As a result, a modified method of misting to wash offthe pollen prior to fertilization is needed to assure crossing orhybridization. Flowers to be used for crossings are selected about 60-90minutes after sunrise. Selection criteria include plants with openflowers, where the stigma has emerged and pollen is visibly attached toa single stigma (there are about 10-20 stigma). Pollen grains are washedoff using 3-4 pumps of water from a spray bottle and with enoughpressure to dislodge the pollen grains without damaging the style.Excess water is then dried off using clean paper towels and about 30minutes later, the styles spring back up and the two lobes of the stigmaare visibly open in a “V” shape. Pollen from another variety or donorparent is then introduced by gently rubbing the stigma and style of thedonor parent to the maternal parent. Most pertinent informationincluding dates and pedigree are then secured to the flowers using tags.

Hybrid vigor has been documented in lettuce and hybrids will be gainingmore and more popularity amongst farmers with uniformity of plantcharacteristics.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possesses the traits tomeet the program goals. The goal is to combine in a single variety orhybrid an improved combination of desirable traits from the parentalgermplasm.

In lettuce, these important traits may include increased head size andweight, higher seed yield, improved color, resistance to diseases andinsects, tolerance to drought and heat, better post-harvest shelf-lifeof the leaves, better standing ability in the field, better uniformity,and better agronomic quality.

In some embodiments, particularly desirable traits that may beincorporated by this disclosure are improved resistance to differentviral, fungal, and bacterial pathogens. Important diseases include butare not limited to fungi such as Bremia lactucae, Fusarium oxysporum,Sclerotinia minor or sclerotorum, Botrytis cinerea, Rhizoctonia solani,Microdochium panattonianum, Verticillium dahliae, Erysiphe cichoracearumor Pythium tracheiphilum, virus, such as LMV (lettuce mosaic virus),TSWV (tomato potted wilt virus), “Big vein” (composed of LBVV (lettucebig vein virus) and MILV (mirafiori lettuce virus)), TBSV (tomato bushystunt virus), LNSV (lettuce necrotic stunt virus), TuMV (turnip mosaicvirus), CMV (cucumber mosaic virus) or BWYV (beet western yellowsvirus), bacteria such as Pseudomonas, Xanthomonas or Rhizomonas.Improved resistance to insect pests is another desirable trait that maybe incorporated into new lettuce plants developed by this disclosure.Insect pests affecting the various species of lettuce include Nasonoviaribisnigri, Myzus persicae, Macrosiphum euphorbiae, Nematodespratylenchus or meloidogyne, leafminers: Liriomyza huidobrensis orPemphigus bursarius.

Other desirable traits include traits related to improved lettuce plantsand parts thereof. A non-limiting list of lettuce phenotypes used duringbreeding selection includes:

Tomato Bushy Stunt (TBSV) resistance or tolerance: TBSV is a viraldisease which causes stunting of growth, leaf mottling, and deformed orabsent heads. When associated with Lettuce Necrotic Stunt Virus (LNSV),another soil born virus, Tomato Bushy Stunt leads to the disease knownas Dieback (Simko et al., 2010. HortScience 45(2): 670-672), resultingin mottling, yellowing, and necrosis of older leaves, stunting of theplant, and eventually death

Tip Burn tolerance: Tip burn tolerance is a tolerance to an abioticdisorder caused by calcium deficiency in growing tissues and resultingin the browning, up to black color, of the margins of young, maturingleaves in head and leaf lettuces. The brown area may be limited to a fewsmall spots at or near the leaf margin, or the entire edge of the leafmay be affected. The term tip burn is usually used to refer to thebrowning in the internal leaves of the plant. Tip burn is also caused byenvironmental conditions that reduce transpiration such as foggyconditions and soil water stress (source: UC Pest Management Guidelines)

Fringe burn tolerance: Fringe burn tolerance is tolerance to browndiscoloration on the outer edge of the lettuce leaf. Fringe burn may belimited to a few spots or cover the entire edge of the leaf. The termFringe burn is usually used to refer to browning on the external leavesof the plant.

Lettuce Breeding

The goal of lettuce breeding is to develop new, unique and superiorlettuce cultivars and hybrids. The breeder initially selects and crossestwo or more parental lines, followed by repeated selfing and selection,producing many new genetic combinations. Another method used to developnew, unique and superior lettuce cultivars occurs when the breederselects and crosses two or more parental lines followed by haploidinduction and chromosome doubling that result in the development ofdihaploid cultivars. The breeder can theoretically generate billions ofdifferent genetic combinations via crossing, selfing and mutations andthe same is true for the utilization of the dihaploid breeding method.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The cultivarsdeveloped are unpredictable. This unpredictability is because thebreeder’s selection occurs in unique environments, with no control atthe DNA level (using conventional breeding procedures or dihaploidbreeding procedures), and with millions of different possible geneticcombinations being generated. A breeder of ordinary skill in the artcannot predict the final resulting cultivars the breeder develops,except possibly in a very gross and general fashion. Thisunpredictability results in the expenditure of large research monies todevelop superior new lettuce cultivars.

The development of commercial lettuce cultivar requires the developmentand selection of lettuce plants, the crossing of these plants, and theevaluation of the crosses.

Pedigree breeding and recurrent selection breeding methods are used todevelop cultivars from breeding populations. Breeding programs combinedesirable traits from two or more cultivars or various broad-basedsources into breeding pools from which cultivars are developed byselfing and selection of desired phenotypes or through the dihaploidbreeding method followed by the selection of desired phenotypes. The newcultivars are evaluated to determine which have commercial potential.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, recurrent selection, andbackcross breeding.

I. Pedigree Selection

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents possessing favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁s or by intercrossing two F₁s (sib mating). The dihaploid breedingmethod could also be used. Selection of the best individuals is usuallybegun in the F₂ population; then, beginning in the F₃, the bestindividuals in the best families are selected. Replicated testing offamilies, or hybrid combinations involving individuals of thesefamilies, often follows in the F₄ generation to improve theeffectiveness of selection for traits with low heritability. At anadvanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease of new cultivars. Similarly, the development of new cultivarsthrough the dihaploid system requires the selection of the cultivarsfollowed by two to five years of testing in replicated plots.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F2 to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F2 individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F2 plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, breeders commonly harvest one or morefruit containing seed from each plant in a population and blend themtogether to form a bulk seed lot. Part of the bulked seed is used toplant the next generation and part is put in reserve. The procedure hasbeen referred to as modified single-seed descent or the bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster than removing one seed from each fruit by handfor the single seed procedure. The multiple-seed procedure also makes itpossible to plant the same number of seeds of a population eachgeneration of inbreeding. Enough seeds are harvested to make up forthose plants that did not germinate or produce seed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., R. W. Allard, 1960, Principles of Plant Breeding, JohnWiley and Son, pp. 115-161; N.W. Simmonds, 1979, Principles of CropImprovement, Longman Group Limited; W. R. Fehr, 1987, Principles of CropDevelopment, Macmillan Publishing Co.; N. F. Jensen, 1988, PlantBreeding Methodology, John Wiley & Sons).

II. Backcross Breeding

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of recurrent parentand the trait of interest from the donor parent are selected andrepeatedly crossed (backcrossed) to the recurrent parent. The resultingplant is expected to have the attributes of the recurrent parent (e.g.,cultivar) and the desirable trait transferred from the donor parent.

When the term lettuce cultivar is used in the context of the presentdisclosure, this also includes any lettuce cultivar plant where one ormore desired trait has been introduced through backcrossing methods,whether such trait is a naturally occurring spontaneous mutation(s), aninduced mutation(s), or a gene or a nucleotide sequence modified by theuse of New Breeding Techniques. Backcrossing methods can be used withthe present disclosure to improve or introduce one or morecharacteristic into the lettuce cultivar of the present disclosure. Theterm “backcrossing” as used herein refers to the repeated crossing of ahybrid progeny back to the recurrent parent, i.e., backcrossing one,two, three, four, five, six, seven, eight, nine, or more times to therecurrent parent. The parental lettuce cultivar plant which contributesthe gene or the genes for the desired characteristic is termed thenonrecurrent or donor parent. This terminology refers to the fact thatthe nonrecurrent parent is used one time in the backcross protocol andtherefore does not recur. The parental lettuce cultivar to which thegene or genes from the nonrecurrent parent are transferred is known asthe recurrent parent as it is used for several rounds in thebackcrossing protocol.

In a typical backcross protocol, the original cultivar of interest(recurrent parent) is crossed to or by a second cultivar (nonrecurrentparent) that carries the gene or genes of interest to be transferred.The resulting progeny from this cross are then crossed again to or bythe recurrent parent and the process is repeated until a lettuce plantis obtained wherein all the desired morphological and physiologicalcharacteristics of the recurrent parent are recovered in the convertedplant, generally determined at a 5% significance level when grown in thesame environmental conditions, in addition to the gene or genestransferred from the nonrecurrent parent. It has to be noted that some,one, two, three or more, self-pollination and growing of populationmight be included between two successive backcrosses. Indeed, anappropriate selection in the population produced by theself-pollination, i.e. selection for the desired trait and physiologicaland morphological characteristics of the recurrent parent might beequivalent to one, two or even three additional backcrosses in acontinuous series without rigorous selection, saving then time, moneyand effort to the breeder. A non-limiting example of such a protocolwould be the following: a) the first generation F1 produced by the crossof the recurrent parent A by the donor parent B is backcrossed to parentA, b) selection is practiced for the plants having the desired trait ofparent B, c) selected plant are self-pollinated to produce a populationof plants where selection is practiced for the plants having the desiredtrait of parent B and physiological and morphological characteristics ofparent A, d) the selected plants are backcrossed one, two, three, four,five, six, seven, eight, nine, or more times to parent A to produceselected backcross progeny plants comprising the desired trait of parentB and the physiological and morphological characteristics of parent A.Step (c) may or may not be repeated and included between the backcrossesof step (d).

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute one or more trait(s) or characteristic(s) in theoriginal inbred parental line in order to find it then in the hybridmade thereof. To accomplish this, a gene or genes of the recurrentinbred is modified or substituted with the desired gene or genes fromthe nonrecurrent parent, while retaining essentially all the rest of thedesired genetic, and therefore the desired physiological andmorphological, constitution of the original inbred. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross; one of the major purposes is to add some commerciallydesirable, agronomically important trait(s) to the plant. The exactbackcrossing protocol will depend on the characteristic(s) or trait(s)being altered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a single gene and dominant allele, multiple genes andrecessive allele(s) may also be transferred and therefore, backcrossbreeding is by no means restricted to character(s) governed by one or afew genes. In fact, the number of genes might be less important that theidentification of the character(s) in the segregating population. Inthis instance it may then be necessary to introduce a test of theprogeny to determine if the desired characteristic(s) has beensuccessfully transferred. Such tests encompass visual inspection, simplecrossing, but also follow up of the characteristic(s) throughgenetically associated markers and molecular assisted breeding tools.For example, selection of progeny containing the transferred trait isdone by direct selection, visual inspection for a trait associated witha dominant allele, while the selection of progeny for a trait that istransferred via a recessive allele, such as the waxy starchcharacteristic in corn, require selfing the progeny or using molecularmarkers to determine which plant carry the recessive allele(s).

Many single gene traits have been identified that are not regularlyselected for in the development of a new lettuce cultivar plantaccording to the disclosure but that can be improved by backcrossingtechniques. Single gene traits may or may not be transgenic. Examples ofthese traits include but are not limited to, male sterility (such as aPR glucanase gene or the ms1, ms2, ms3, ms4, ms5, ms7 genes), herbicideresistance (such as bar or PAT genes). Gene controlling resistance tothe lettuce leaf aphid Nasonovia ribisnigri (Nr gene) can be found inVan der Arend and Schijndel in Breeding for Resistance to insects andMites, IOBC wprs Bulletin 22(10), 35-43 (1999). Other traits forresistance or tolerance to an infection by a virus, a bacterium, aninsect or a fungus, might be obtained from the genes for resistance toBremia Dm10, R17, Dm5, Dm8, R36, R37 (genes located on cluster 1 ofLactuca sativa), Dm1, Dm2, Dm3, Dm6, Dm14, Dm15, Dm16, Dm18 (geneslocated on cluster 2 of Lactuca sativa), Dm4, Dm7, Dm11, R38 (geneslocated on cluster 4 of Lactuca sativa); or the Tu gene for resistanceto TuMV located on cluster 1; or from the genes mol.1 and mol.2 forresistance to LMV located on cluster 4. Clusters 1, 2 and 4 cited abovehave been defined by Michelmore R. W. (Plant Pathol, 1987, vol. 36, no4: 499-514 [4], Theor. Appl. Genet., 1993, vol. 85, No 8: 985-993. Thesegenes are generally inherited through the nucleus. Some other singlegene traits are described in U.S. Pat. Nos. 5,777,196, 5,948,957, and5,969,212, the disclosures of which are specifically hereby incorporatedby reference.

The backcross breeding method provides a precise way of improvingvarieties that excel in a large number of attributes but are deficientin a few characteristics. (Page 150 of the Pr. R.W. Allard’s 1960 book,published by John Wiley & Sons, Inc, Principles of Plant Breeding). Themethod makes use of a series of backcrosses to the variety to beimproved during which the character or the characters in whichimprovement is sought is maintained by selection. At the end of thebackcrossing the gene or genes being transferred unlike all other genes,will be heterozygous. Selfing after the last backcross produceshomozygosity for this gene pair(s) and, coupled with selection, willresult in a parental line of a hybrid variety with exactly oressentially the same adaptation, yielding ability and qualitycharacteristics of the recurrent parent but superior to that parent inthe particular characteristic(s) for which the improvement program wasundertaken. Therefore, this method provides the plant breeder with ahigh degree of genetic control of this work.

The method is scientifically exact because the morphological andagricultural features of the improved variety could be described inadvance and because a similar variety could, if it were desired, be breda second time by retracing the same steps (Briggs, “Breeding wheatsresistant to bunt by the backcross method”, 1930 Jour. Amer. Soc.Agron., 22: 289-244).

Backcrossing is a powerful mechanism for achieving homozygosity and anypopulation obtained by backcrossing must rapidly converge on thegenotype of the recurrent parent. When backcrossing is made the basis ofa plant breeding program, the genotype of the recurrent parent will betheoretically modified only with regards to genes being transferred,which are maintained in the population by selection.

Successful backcrosses are, for example, the transfer of stem rustresistance from ‘Hope’ wheat to ‘Bart wheat’ and even pursuing thebackcrosses with the transfer of bunt resistance to create ‘Bart 38’,having both resistances. Also highlighted by Allard is the successfultransfer of mildew, leaf spot and wilt resistances in California Commonalfalfa to create ‘Caliverde’. This new ‘Caliverde’ variety producedthrough the backcross process is indistinguishable from CaliforniaCommon except for its resistance to the three named diseases.

One of the advantages of the backcross method is that the breedingprogram can be carried out in almost every environment that will allowthe development of the character being transferred or when usingmolecular markers that can identify the trait of interest.

The backcross technique is not only desirable when breeding for diseaseresistance but also for the adjustment of morphological characters,color characteristics and simply inherited quantitative characters suchas earliness, plant height and seed size and shape.

III. Open-Pollinated Populations

The improvement of open-pollinated populations of such crops as rye,maize and sugar beets, herbage grasses, legumes such as alfalfa andclover, and tropical tree crops such as cacao, coconuts, oil palm andsome rubber, depends essentially upon changing gene-frequencies towardsfixation of favorable alleles while maintaining a high (but far frommaximal) degree of heterozygosity.

Uniformity in such populations is impossible and trueness-to-type in anopen-pollinated variety is a statistical feature of the population as awhole, not a characteristic of individual plants. Thus, theheterogeneity of open-pollinated populations contrasts with thehomogeneity (or virtually so) of inbred lines, clones and hybrids.

Population improvement methods fall naturally into two groups, thosebased on purely phenotypic selection, normally called mass selection,and those based on selection with progeny testing. Interpopulationimprovement utilizes the concept of open breeding populations; allowinggenes to flow from one population to another. Plants in one population(cultivar, strain, ecotype, or any germplasm source) are crossed eithernaturally (e.g., by wind) or by hand or by bees (commonly Apis melliferaL. or Megachile rotundata F.) with plants from other populations.Selection is applied to improve one (or sometimes both) population(s) byisolating plants with desirable traits from both sources.

There are basically two primary methods of open-pollinated populationimprovement.

First, there is the situation in which a population is changed en masseby a chosen selection procedure. The outcome is an improved populationthat is indefinitely propagable by random-mating within itself inisolation.

Second, the synthetic variety attains the same end result as populationimprovement, but is not itself propagable as such; it has to bereconstructed from parental lines or clones. These plant breedingprocedures for improving open-pollinated populations are well known tothose skilled in the art and comprehensive reviews of breedingprocedures routinely used for improving cross-pollinated plants areprovided in numerous texts and articles, including: Allard, Principlesof Plant Breeding, John Wiley & Sons, Inc. (1960); Simmonds, Principlesof Crop Improvement, Longman Group Limited (1979); Hallauer and Miranda,Quantitative Genetics in Maize Breeding, Iowa State University Press(1981); and, Jensen, Plant Breeding Methodology, John Wiley & Sons, Inc.(1988).

A) Mass Selection

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued. In massselection, desirable individual plants are chosen, harvested, and theseed composited without progeny testing to produce the followinggeneration. Since selection is based on the maternal parent only, andthere is no control over pollination, mass selection amounts to a formof random mating with selection. As stated above, the purpose of massselection is to increase the proportion of superior genotypes in thepopulation.

B) Synthetics

A synthetic variety is produced by intercrossing a number of genotypesselected for good combining ability in all possible hybrid combinations,with subsequent maintenance of the variety by open pollination. Whetherparents are (more or less inbred) seed-propagated lines, as in somesugar beet and beans (Vicia) or clones, as in herbage grasses, cloversand alfalfa, makes no difference in principle. Parents are selected ongeneral combining ability, sometimes by test crosses or topcrosses, moregenerally by polycrosses. Parental seed lines may be deliberately inbred(e.g. by selfing or sib crossing). However, even if the parents are notdeliberately inbred, selection within lines during line maintenance willensure that some inbreeding occurs. Clonal parents will, of course,remain unchanged and highly heterozygous.

Whether a synthetic can go straight from the parental seed productionplot to the farmer or must first undergo one or more cycles ofmultiplication depends on seed production and the scale of demand forseed. In practice, grasses and clovers are generally multiplied once ortwice and are thus considerably removed from the original synthetic.

While mass selection is sometimes used, progeny testing is generallypreferred for polycrosses, because of their operational simplicity andobvious relevance to the objective, namely exploitation of generalcombining ability in a synthetic.

The number of parental lines or clones that enters a synthetic varieswidely. In practice, numbers of parental lines range from 10 to severalhundred, with 100-200 being the average. Broad based synthetics formedfrom 100 or more clones would be expected to be more stable during seedmultiplication than narrow based synthetics.

IV. Hybrids

A hybrid is an individual plant resulting from a cross between parentsof differing genotypes. Commercial hybrids are now used extensively inmany crops, including corn (maize), sorghum, sugarbeet, sunflower andbroccoli as well as leafy vegetables such as lettuce. Hybrids can beformed in a number of different ways, including by crossing two parentsdirectly (single cross hybrids), by crossing a single cross hybrid withanother parent (three-way or triple cross hybrids), or by crossing twodifferent hybrids (four-way or double cross hybrids).

Strictly speaking, most individuals in an out breeding (i.e.,open-pollinated) population are hybrids, but the term is usuallyreserved for cases in which the parents are individuals whose genomesare sufficiently distinct for them to be recognized as different speciesor subspecies. Hybrids may be fertile or sterile depending onqualitative and/or quantitative differences in the genomes of the twoparents. Heterosis, or hybrid vigor, is usually associated withincreased heterozygosity that results in increased vigor of growth,survival, and fertility of hybrids as compared with the parental linesthat were used to form the hybrid. Maximum heterosis is usually achievedby crossing two genetically different, highly inbred lines.

Hybrid commercial lettuce seed can be produced by insect pollination,see U.S. Pat. 8,716,551 which is specifically hereby incorporated byreference.

Once the inbreds that give the best hybrid performance have beenidentified, the hybrid seed can be reproduced indefinitely as long asthe homogeneity of the inbred parent is maintained. A single-crosshybrid is produced when two inbred lines are crossed to produce the F1progeny. A double-cross hybrid is produced from four inbred linescrossed in pairs (AxB and CxD) and then the two F1 hybrids are crossedagain (AxB) x (CxD). Much of the hybrid vigor and uniformity exhibitedby F1 hybrids is lost in the next generation (F2). Consequently, seedfrom F2 hybrid varieties is not used for planting stock.

The production of hybrids is a well-developed industry, involving theisolated production of both the parental lines and the hybrids whichresult from crossing those lines. For a detailed discussion of thehybrid production process, see, e.g., Wright, Commercial Hybrid SeedProduction 8:161-176, In Hybridization of Crop Plants.

V. Bulk Segregation Analysis (BSA)

BSA, a.k.a. bulked segregation analysis, or bulk segregant analysis, isa method described by Michelmore et al. (Michelmore et al., 1991,Identification of markers linked to disease-resistance genes by bulkedsegregant analysis: a rapid method to detect markers in specific genomicregions by using segregating populations. Proceedings of the NationalAcademy of Sciences, USA, 99:9828-9832) and Quarrie et al. (Quarrie etal., 1999, Journal of Experimental Botany, 50(337):1299-1306).

For BSA of a trait of interest, parental lines with certain differentphenotypes are chosen and crossed to generate F2, doubled haploid orrecombinant inbred populations with QTL analysis. The population is thenphenotyped to identify individual plants or lines having high or lowexpression of the trait. Two DNA bulks are prepared, one from theindividuals having one phenotype (e.g., resistant to virus), and theother from the individuals having reversed phenotype (e.g., susceptibleto virus), and analyzed for allele frequency with molecular markers.Only a few individuals are required in each bulk (e.g., 10 plants each)if the markers are dominant (e.g., RAPDs). More individuals are neededwhen markers are co-dominant (e.g., RFLPs, SNPs or SSRs). Markers linkedto the phenotype can be identified and used for breeding or QTL mapping.

VI. Hand-Pollination Method

Hand pollination describes the crossing of plants via the deliberatefertilization of female ovules with pollen from a desired male parentplant. In some embodiments the donor or recipient female parent and thedonor or recipient male parent line are planted in the same field. Insome embodiments the donor or recipient female parent and the donor orrecipient male parent line are planted in the same greenhouse. Theinbred male parent can be planted earlier than the female parent toensure adequate pollen supply at the pollination time. In someembodiments, the male parent and female parent can be planted at a ratioof 1 male parent to 4-10 female parents. The male parent may be plantedat the top of the field for efficient male flower collection duringpollination. Pollination is started when the female parent flower isready to be fertilized. Female flower buds that are ready to open in thefollowing days are identified, covered with paper cups or small paperbags that prevent bee or any other insect from visiting the femaleflowers, and marked with any kind of material that can be easily seenthe next morning. In some embodiments, this process is best done in theafternoon. The male flowers of the male parent are collected in theearly morning before they are open and visited by pollinating insects.The covered female flowers of the female parent, which have opened, areun-covered and pollinated with the collected fresh male flowers of themale parent, starting as soon as the male flower sheds pollen. Thepollinated female flowers are again covered after pollination to preventbees and any other insects visit. The pollinated female flowers are alsomarked. The marked flowers are harvested. In some embodiments, the malepollen used for fertilization has been previously collected and stored.

VII. Bee-Pollination Method

Using the bee-pollination method, the parent plants are usually plantedwithin close proximity. In some embodiments more female plants areplanted to allow for a greater production of seed. Insects are placed inthe field or greenhouses for transfer of pollen from the male parent tothe female flowers of the female parent.

VIII. Targeting Induced Local Lesions in Genomes (TILLING)

Breeding schemes of the present application can include crosses withTILLING® plant cultivars. TILLING® is a method in molecular biology thatallows directed identification of mutations in a specific gene. TILLING®was introduced in 2000, using the model plant Arabidopsis thaliana.TILLING® has since been used as a reverse genetics method in otherorganisms such as zebrafish, corn, wheat, rice, soybean, tomato andlettuce.

The method combines a standard and efficient technique of mutagenesiswith a chemical mutagen (e.g., Ethyl methanesulfonate (EMS)) with asensitive DNA screening-technique that identifies single base mutations(also called point mutations) in a target gene. EcoTILLING is a methodthat uses TILLING® techniques to look for natural mutations inindividuals, usually for population genetics analysis (see Comai, etal., 2003 The Plant Journal 37, 778-786; Gilchrist et al. 2006 Mol.Ecol. 15, 1367-1378; Mejlhede et al. 2006 Plant Breeding 125, 461-467;Nieto et al. 2007 BMC Plant Biology 7, 34-42, each of which isincorporated by reference hereby for all purposes). DEcoTILLING is amodification of TILLING® and EcoTILLING which uses an inexpensive methodto identify fragments (Garvin et al., 2007, DEco-TILLING: An inexpensivemethod for SNP discovery that reduces ascertainment bias. MolecularEcology Notes 7, 735-746).

The TILLING® method relies on the formation of heteroduplexes that areformed when multiple alleles (which could be from a heterozygote or apool of multiple homozygotes and heterozygotes) are amplified in a PCR,heated, and then slowly cooled. As DNA bases are not pairing at themismatch of the two DNA strands (the induced mutation in TILLING® or thenatural mutation or SNP in EcoTILLING), they provoke a shape change inthe double strand DNA fragment which is then cleaved by single strandednucleases. The products are then separated by size on several differentplatforms.

Several TILLING® centers exists over the world that focus onagriculturally important species: UC Davis (USA), focusing on Rice;Purdue University (USA), focusing on Maize; University of BritishColumbia (CA), focusing on Brassica napus; John Innes Centre (UK),focusing on Brassica rapa; Fred Hutchinson Cancer Research, focusing onArabidopsis; Southern Illinois University (USA), focusing on Soybean;John Innes Centre (UK), focusing on Lotus and Medicago ; and INRA(France), focusing on Pea and Tomato.

More detailed description on methods and compositions on TILLING® can befound in US 5994075, US 2004/0053236 A1, WO 2005/055704, and WO2005/048692, each of which is hereby incorporated by reference for allpurposes.

Thus in some embodiments, the breeding methods of the present disclosureinclude breeding with one or more TILLING plant lines with one or moreidentified mutations.

IX. Mutation Breeding

Mutation breeding is another method of introducing new variation andsubsequent traits into lettuce plants. Mutations that occurspontaneously or are artificially induced can be useful sources ofvariability for a plant breeder. The goal of artificial mutagenesis isto increase the rate of mutation for a desired characteristic. Mutationrates can be increased by many different means or mutating agentsincluding temperature, long-term seed storage, tissue cultureconditions, radiation (such as X-rays, Gamma rays, neutrons, Betaradiation, or ultraviolet radiation), chemical mutagens (such as baseanalogs like 5-bromo-uracil), antibiotics, alkylating agents (such assulfur mustards, nitrogen mustards, epoxides, ethyleneamines, sulfates,sulfonates, sulfones, or lactones), azide, hydroxylamine, nitrous acidor acridines. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques. Details of mutation breeding can be found in W. R.Fehr, 1993, Principles of Cultivar Development, Macmillan Publishing Co.

New breeding techniques such as the ones involving the uses ofengineered nuclease to enhance the efficacy and precision of geneediting in combination with oligonucleotides including, but not limitedto Zinc Finger Nucleases (ZFN), TAL effector nucleases (TALENs) andclustered regularly interspaced short palindromic repeats(CRISPR)-associated endonuclease Cas9 (CRISPR-Cas9) using such as Cas9,Cas12a/Cpf1, Cas13/C2c2, CasX and CasY or oligonucleotide directedmutagenesis shall also be used to generate genetic variability andintroduce new traits into lettuce varieties.

X. Double Haploids and Chromosome Doubling

One way to obtain homozygous plants without the need to cross twoparental lines followed by a long selection of the segregating progeny,and/or multiple backcrossings is to produce haploids and then double thechromosomes to form doubled haploids. Haploid plants can occurspontaneously, or may be artificially induced via chemical treatments orby crossing plants with inducer lines (Seymour et al. 2012, PNAS vol109, pg 4227-4232; Zhang et al., 2008 Plant Cell Rep. Dec 27(12)1851-60). The production of haploid progeny can occur via a variety ofmechanisms which can affect the distribution of chromosomes duringgamete formation. The chromosome complements of haploids sometimesdouble spontaneously to produce homozygous doubled haploids (DHs).Mixoploids, which are plants which contain cells having differentploidies, can sometimes arise and may represent plants that areundergoing chromosome doubling so as to spontaneously produce doubledhaploid tissues, organs, shoots, floral parts or plants. Another commontechnique is to induce the formation of double haploid plants with achromosome doubling treatment such as colchicine (El-Hennawy et al.,2011 Vol 56, issue 2 pg 63-72; Doubled Haploid Production in Crop Plants2003 edited by Maluszynski ISBN 1-4020-1544-5). The production ofdoubled haploid plants yields highly uniform cultivars and is especiallydesirable as an alternative to sexual inbreeding of longer-generationcrops. By producing doubled haploid progeny, the number of possible genecombinations for inherited traits is more manageable. Thus, an efficientdoubled haploid technology can significantly reduce the time and thecost of inbred and cultivar development.

XI. Protoplast Fusion

In another method for breeding plants, protoplast fusion can also beused for the transfer of trait-conferring genomic material from a donorplant to a recipient plant. Protoplast fusion is an induced orspontaneous union, such as a somatic hybridization, between two or moreprotoplasts (cells of which the cell walls are removed by enzymatictreatment) to produce a single bi- or multi-nucleate cell. The fusedcell that may even be obtained with plant species that cannot beinterbred in nature is tissue cultured into a hybrid plant exhibitingthe desirable combination of traits.

XII. Embryo Rescue

Alternatively, embryo rescue may be employed in the transfer ofresistance-conferring genomic material from a donor plant to a recipientplant. Embryo rescue can be used as a procedure to isolate embryos fromcrosses to rapidly move to the next generation of backcrossing orselfing or wherein plants fail to produce viable seed. In this process,the fertilized ovary or immature seed of a plant is tissue cultured tocreate new plants (see Pierik, 1999, In Vitro Culture of Higher Plants,Springer, ISBN 079235267, 9780792352679, which is incorporated herein byreference in its entirety).

Breeding Evaluation

Each breeding program can include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested per se and inhybrid combination and compared to appropriate standards in environmentsrepresentative of the commercial target area(s). The best lines arecandidates for use as parents in new commercial cultivars; those stilldeficient in a few traits may be used as parents to produce newpopulations for further selection or in a backcross program to improvethe parent lines for a specific trait.

In some embodiments, the plants are selected on the basis of one or morephenotypic traits. Skilled persons will readily appreciate that suchtraits include any observable characteristic of the plant, including forexample growth rate, vigor, plant health, maturity, plant height, leafcoverage, weight, total yield, color, taste, sugar levels, aroma, smell,changes in the production of one or more compounds by the plant(including for example, metabolites, proteins, drugs, carbohydrates,oils, and any other compounds).

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

It should be appreciated that in certain embodiments, plants may beselected based on the absence, suppression or inhibition of a certainfeature or trait (such as an undesirable feature or trait) as opposed tothe presence of a certain feature or trait (such as a desirable featureor trait).

Selecting plants based on genotypic information is also envisaged (forexample, including the pattern of plant gene expression, genotype, orpresence of genetic markers). Where the presence of one or more geneticmarker is assessed, the one or more marker may already be known and/orassociated with a particular characteristic of a plant; for example, amarker or markers may be associated with an increased growth rate ormetabolite profile. This information could be used in combination withassessment based on other characteristics in a method of the disclosureto select for a combination of different plant characteristics that maybe desirable. Such techniques may be used to identify novel quantitativetrait loci (QTLs). By way of example, plants may be selected based ongrowth rate, size (including but not limited to weight, height, leafsize, stem size, branching pattern, or the size of any part of theplant), general health, survival, tolerance to adverse physicalenvironments and/or any other characteristic, as described hereinbefore.

Further non-limiting examples include selecting plants based on: speedof seed germination; quantity of biomass produced; increased root,and/or leaf/shoot growth that leads to an increased yield (herbage orgrain or fiber or oil) or biomass production; effects on plant growththat results in an increased seed yield for a crop; effects on plantgrowth which result in an increased head yield; effects on plant growththat lead to an increased resistance or tolerance to disease includingfungal, viral or bacterial diseases, to mycoplasma, or to pests such asinsects, mites or nematodes in which damage is measured by decreasedfoliar symptoms such as the incidence of bacterial or fungal lesions, orarea of damaged foliage or reduction in the numbers of nematode cysts orgalls on plant roots, or improvements in plant yield in the presence ofsuch plant pests and diseases; effects on plant growth that lead toincreased metabolite yields; effects on plant growth that lead toimproved aesthetic appeal which may be particularly important in plantsgrown for their form, color or taste, for example the color intensity oflettuce leaves, or the taste of said leaves.

Molecular Breeding Evaluation Techniques

Selection of plants based on phenotypic or genotypic information may beperformed using techniques such as, but not limited to: high through-putscreening of chemical components of plant origin, sequencing techniquesincluding high through-put sequencing of genetic material, differentialdisplay techniques (including DDRT-PCR, and DD-PCR), nucleic acidmicroarray techniques, RNA-seq (Transcriptome Sequencing), qRTPCR(quantitative real time PCR).

In one embodiment, the evaluating step of a plant breeding programinvolves the identification of desirable traits in progeny plants.Progeny plants can be grown in, or exposed to conditions designed toemphasize a particular trait (e.g. drought conditions for droughttolerance, lower temperatures for freezing tolerant traits). Progenyplants with the highest scores for a particular trait may be used forsubsequent breeding steps.

In some embodiments, plants selected from the evaluation step canexhibit a 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120% or more improvement in aparticular plant trait compared to a control plant.

In other embodiments, the evaluating step of plant breeding comprisesone or more molecular biological tests for genes or other markers. Forexample, the molecular biological test can involve probe hybridizationand/or amplification of nucleic acid (e.g., measuring nucleic aciddensity by Northern or Southern hybridization, PCR) and/or immunologicaldetection (e.g., measuring protein density, such as precipitation andagglutination tests, ELISA (e.g., Lateral Flow test or DAS-ELISA),Western blot, Radioimmune Assay (RIA), immune labeling, immunosorbentelectron microscopy (ISEM), and/or dot blot).

The procedure to perform a nucleic acid hybridization, an amplificationof nucleic acid (e.g., PCR, RT-PCR) or an immunological detection (e.g.,precipitation and agglutination tests, ELISA (e.g., Lateral Flow test orDAS-ELISA), Western blot, RIA, immunogold or immunofluorescent labeling,immunosorbent electron microscopy (ISEM), and/or dot blot tests) areperformed as described elsewhere herein and well-known by one skilled inthe art.

In one embodiment, the evaluating step comprises PCR (semi-quantitativeor quantitative), wherein primers are used to amplify one or morenucleic acid sequences of a desirable gene, or a nucleic acid associatedwith said gene, or QTL or a desirable trait (e.g., a co-segregatingnucleic acid, or other marker).

In another embodiment, the evaluating step comprises immunologicaldetection (e.g., precipitation and agglutination tests, ELISA (e.g.,Lateral Flow test or DAS-ELISA), Western blot, RIA, immuno labeling(gold, fluorescent, or other detectable marker), immunosorbent electronmicroscopy (ISEM), and/or dot blot), wherein one or more gene ormarker-specific antibodies are used to detect one or more desirableproteins. In one embodiment, said specific antibody is selected from thegroup consisting of polyclonal antibodies, monoclonal antibodies,antibody fragments, and combination thereof.

Reverse Transcription Polymerase Chain Reaction (RT-PCR) can be utilizedin the present disclosure to determine expression of a gene to assistduring the selection step of a breeding scheme. It is a variant ofpolymerase chain reaction (PCR), a laboratory technique commonly used inmolecular biology to generate many copies of a DNA sequence, a processtermed “amplification”. In RT-PCR, however, RNA strand is first reversetranscribed into its DNA complement (complementary DNA, or cDNA) usingthe enzyme reverse transcriptase, and the resulting cDNA is amplifiedusing traditional or real-time PCR.

RT-PCR utilizes a pair of primers, which are complementary to a definedsequence on each of the two strands of the cDNA. These primers are thenextended by a DNA polymerase and a copy of the strand is made after eachcycle, leading to logarithmic amplification.

RT-PCR includes three major steps. The first step is the reversetranscription (RT) where RNA is reverse transcribed to cDNA using areverse transcriptase and primers. This step is very important in orderto allow the performance of PCR since DNA polymerase can act only on DNAtemplates. The RT step can be performed either in the same tube with PCR(one-step PCR) or in a separate one (two-step PCR) using a temperaturebetween 40° C. and 60° C., depending on the properties of the reversetranscriptase used.

The next step involves the denaturation of the dsDNA at 95° C., so thatthe two strands separate and the primers can bind again at lowertemperatures and begin a new chain reaction. Then, the temperature isdecreased until it reaches the annealing temperature which can varydepending on the set of primers used, their concentration, the probe andits concentration (if used), and the cation concentration. The mainconsideration, of course, when choosing the optimal annealingtemperature is the melting temperature (Tm) of the primers and probes(if used). The annealing temperature chosen for a PCR depends directlyon length and composition of the primers. This is the result of thedifference of hydrogen bonds between A-T (2 bonds) and G-C (3 bonds). Anannealing temperature about 5 degrees below the lowest Tm of the pair ofprimers is usually used.

The final step of PCR amplification is the DNA extension from theprimers which is done by the thermostable Taq DNA polymerase usually at72° C., which is the optimal temperature for the polymerase to work. Thelength of the incubation at each temperature, the temperaturealterations and the number of cycles are controlled by a programmablethermal cycler. The analysis of the PCR products depends on the type ofPCR applied. If a conventional PCR is used, the PCR product is detectedusing for example agarose gel electrophoresis or other polymer gel likepolyacrylamide gels and ethidium bromide (or other nucleic acidstaining).

Conventional RT-PCR is a time-consuming technique with importantlimitations when compared to real time PCR techniques. This, combinedwith the fact that ethidium bromide has low sensitivity, yields resultsthat are not always reliable. Moreover, there is an increasedcross-contamination risk of the samples since detection of the PCRproduct requires the post-amplification processing of the samples.Furthermore, the specificity of the assay is mainly determined by theprimers, which can give false-positive results. However, the mostimportant issue concerning conventional RT-PCR is the fact that it is asemi or even a low quantitative technique, where the amplicon can bevisualized only after the amplification ends.

Real time RT-PCR provides a method where the amplicons can be visualizedas the amplification progresses using a fluorescent reporter molecule.There are three major kinds of fluorescent reporters used in real timeRT-PCR, general nonspecific DNA Binding Dyes such as SYBR Green I,TaqMan Probes and Molecular Beacons (including Scorpions).

The real time PCR thermal cycler has a fluorescence detection threshold,below which it cannot discriminate the difference between amplificationgenerated signal and background noise. On the other hand, thefluorescence increases as the amplification progresses and theinstrument performs data acquisition during the annealing step of eachcycle. The number of amplicons will reach the detection baseline after aspecific cycle, which depends on the initial concentration of the targetDNA sequence. The cycle at which the instrument can discriminate theamplification generated fluorescence from the background noise is calledthe threshold cycle (Ct). The higher is the initial DNA concentration,the lower its Ct will be.

Other forms of nucleic acid detection can include next generationsequencing methods such as DNA SEQ or RNA SEQ using any known sequencingplatform including, but not limited to: Roche 454, Solexa GenomeAnalyzer, AB SOLiD, Illumina GA/HiSeq, Ion PGM, Mi Seq, among others(Liu et al,. 2012 Journal of Biomedicine and Biotechnology Volume 2012ID 251364; Franca et al., 2002 Quarterly Reviews of Biophysics 35 pg169-200; Mardis 2008 Genomics and Human Genetics vol 9 pg 387-402).

In other embodiments, nucleic acids may be detected with other highthroughput hybridization technologies including microarrays, gene chips,LNA probes, nanoStrings, and fluorescence polarization detection amongothers.

In some embodiments, detection of markers can be achieved at an earlystage of plant growth by harvesting a small tissue sample (e.g., branch,or leaf disk). This approach is preferable when working with largepopulations as it allows breeders to weed out undesirable progeny at anearly stage and conserve growth space and resources for progeny whichshow more promise. In some embodiments the detection of markers isautomated, such that the detection and storage of marker data is handledby a machine. Recent advances in robotics have also led to full serviceanalysis tools capable of handling nucleic acid/protein markerextractions, detection, storage and analysis.

Quantitative Trait Loci

Breeding schemes of the present application can include crosses betweendonor and recipient plants. In some embodiments, said donor plantscontain a gene or genes of interest which may confer the plant with adesirable phenotype. The recipient line can be an elite line or cultivarhaving certain favorite traits for commercial production. In oneembodiment, the elite line may contain other genes that also impart saidline with the desired phenotype. When crossed together, the donor andrecipient plant may create a progeny plant with combined desirable lociwhich may provide quantitatively additive effect of a particularcharacteristic. In that case, QTL mapping can be involved to facilitatethe breeding process.

A QTL (quantitative trait locus) mapping can be applied to determine theparts of the donor plant’s genome conferring the desirable phenotype,and facilitate the breeding methods. Inheritance of quantitative traitsor polygenic inheritance refers to the inheritance of a phenotypiccharacteristic that varies in degree and can be attributed to theinteractions between two or more genes and their environment. Though notnecessarily genes themselves, quantitative trait loci (QTLs) arestretches of DNA that are closely linked to the genes that underlie thetrait in question. QTLs can be molecularly identified to help mapregions of the genome that contain genes involved in specifying aquantitative trait. This can be an early step in identifying andsequencing these genes.

Typically, QTLs underlie continuous traits (those traits that varycontinuously, e.g. yield, height, level of resistance to virus, etc.) asopposed to discrete traits (traits that have two or several charactervalues, e.g. smooth vs. wrinkled peas used by Mendel in hisexperiments). Moreover, a single phenotypic trait is usually determinedby many genes. Consequently, many QTLs are associated with a singletrait.

A quantitative trait locus (QTL) is a region of DNA that is associatedwith a particular phenotypic trait. Knowing the number of QTLs thatexplains variation in the phenotypic trait tells about the geneticarchitecture of a trait. It may tell that a trait is controlled by manygenes of small effect, or by a few genes of large effect or by a severalgenes of small effect and few genes of larger effect.

Another use of QTLs is to identify candidate genes underlying a trait.Once a region of DNA is identified as contributing to a phenotype, itcan be sequenced. The DNA sequence of any genes in this region can thenbe compared to a database of DNA for genes whose function is alreadyknown.

In a recent development, classical QTL analyses are combined with geneexpression profiling i.e. by DNA microarrays. Such expression QTLs(e-QTLs) describes cis- and trans-controlling elements for theexpression of often disease-associated genes. Observed epistatic effectshave been found beneficial to identify the gene responsible by across-validation of genes within the interacting loci with metabolicpathway and scientific literature databases.

QTL mapping is the statistical study of the alleles that occur in alocus and the phenotypes (physical forms or traits) that they produce(see, Meksem and Kahl, The handbook of plant genome mapping: genetic andphysical mapping, 2005, Wiley-VCH, ISBN 3527311165, 9783527311163).Because most traits of interest are governed by more than one gene,defining and studying the entire locus of genes related to a trait giveshope of understanding what effect the genotype of an individual mighthave in the real world.

Statistical analysis is required to demonstrate that different genesinteract with one another and to determine whether they produce asignificant effect on the phenotype. QTLs identify a particular regionof the genome as containing one or several genes, i.e. a cluster ofgenes that are associated with the trait being assayed or measured. Theyare shown as intervals across a chromosome, where the probability ofassociation is plotted for each marker used in the mapping experiment.

To begin, a set of genetic markers must be developed for the species inquestion. A marker is an identifiable region of variable DNA. Biologistsare interested in understanding the genetic basis of phenotypes(physical traits). The aim is to find a marker that is significantlymore likely to co-occur with the trait than expected by chance, that is,a marker that has a statistical association with the trait. Ideally,they would be able to find the specific gene or genes in question, butthis is a long and difficult undertaking. Instead, they can more readilyfind regions of DNA that are very close to the genes in question. When aQTL is found, it is often not the actual gene underlying the phenotypictrait, but rather a region of DNA that is closely linked with the gene.

For organisms whose genomes are known, one might now try to excludegenes in the identified region whose function is known with somecertainty not to be connected with the trait in question. If the genomeis not available, it may be an option to sequence the identified regionand determine the putative functions of genes by their similarity togenes with known function, usually in other genomes. This can be doneusing BLAST, an online tool that allows users to enter a primarysequence and search for similar sequences within the BLAST database ofgenes from various organisms.

Another interest of statistical geneticists using QTL mapping is todetermine the complexity of the genetic architecture underlying aphenotypic trait. For example, they may be interested in knowing whethera phenotype is shaped by many independent loci, or by a few loci, andhow those loci interact. This can provide information on how thephenotype may be evolving.

Molecular markers are used for the visualization of differences innucleic acid sequences. This visualization is possible due to DNA-DNAhybridization techniques (RFLP) and/or due to techniques using thepolymerase chain reaction (e.g. STS, SNPs, microsatellites, AFLP). Alldifferences between two parental genotypes will segregate in a mappingpopulation based on the cross of these parental genotypes. Thesegregation of the different markers may be compared and recombinationfrequencies can be calculated. The recombination frequencies ofmolecular markers on different chromosomes are generally 50%. Betweenmolecular markers located on the same chromosome the recombinationfrequency depends on the distance between the markers. A lowrecombination frequency usually corresponds to a low distance betweenmarkers on a chromosome. Comparing all recombination frequencies willresult in the most logical order of the molecular markers on thechromosomes. This most logical order can be depicted in a linkage map(Paterson, 1996, Genome Mapping in Plants. R.G. Landes, Austin.). Agroup of adjacent or contiguous markers on the linkage map that isassociated to a reduced disease incidence and/or a reduced lesion growthrate pinpoints the position of a QTL.

The nucleic acid sequence of a QTL may be determined by methods known tothe skilled person. For instance, a nucleic acid sequence comprisingsaid QTL or a resistance-conferring part thereof may be isolated from adonor plant by fragmenting the genome of said plant and selecting thosefragments harboring one or more markers indicative of said QTL.Subsequently, or alternatively, the marker sequences (or parts thereof)indicative of said QTL may be used as (PCR) amplification primers, inorder to amplify a nucleic acid sequence comprising said QTL from agenomic nucleic acid sample or a genome fragment obtained from saidplant. The amplified sequence may then be purified in order to obtainthe isolated QTL. The nucleotide sequence of the QTL, and/or of anyadditional markers comprised therein, may then be obtained by standardsequencing methods.

One or more such QTLs associated with a desirable trait in a donor plantcan be transferred to a recipient plant to incorporate the desirabletrait into progeny plants by transferring and/or breeding methods.

In one embodiment, an advanced backcross QTL analysis (AB-QTL) is usedto discover the nucleotide sequence or the QTLs responsible for theresistance of a plant. Such method was proposed by Tanksley and Nelsonin 1996 (Tanksley and Nelson, 1996, Advanced backcross QTL analysis: amethod for simultaneous discovery and transfer of valuable QTL fromun-adapted germplasm into elite breeding lines. Theor Appl Genet92:191-203) as a new breeding method that integrates the process of QTLdiscovery with variety development, by simultaneously identifying andtransferring useful QTL alleles from un-adapted (e.g., land races, wildspecies) to elite germplasm, thus broadening the genetic diversityavailable for breeding. AB-QTL strategy was initially developed andtested in tomato, and has been adapted for use in other crops includingrice, maize, wheat, pepper, barley, and bean. Once favorable QTL allelesare detected, only a few additional marker-assisted generations arerequired to generate near isogenic lines (NILs) or introgression lines(ILs) that can be field tested in order to confirm the QTL effect andsubsequently used for variety development.

Isogenic lines in which favorable QTL alleles have been fixed can begenerated by systematic backcrossing and introgressing of marker-defineddonor segments in the recurrent parent background. These isogenic linesare referred to as near isogenic lines (NILs), introgression lines(ILs), backcross inbred lines (BILs), backcross recombinant inbred lines(BCRIL), recombinant chromosome substitution lines (RCSLs), chromosomesegment substitution lines (CSSLs), and stepped aligned inbredrecombinant strains (STAIRSs). An introgression line in plant molecularbiology is a line of a crop species that contains genetic materialderived from a similar species. ILs represent NILs with relatively largeaverage introgression length, while BILs and BCRILs are backcrosspopulations generally containing multiple donor introgressions per line.As used herein, the term “introgression lines or ILs” refers to plantlines containing a single marker defined homozygous donor segment, andthe term “pre-ILs” refers to lines which still contain multiplehomozygous and/or heterozygous donor segments.

To enhance the rate of progress of introgression breeding, a geneticinfrastructure of exotic libraries can be developed. Such an exoticlibrary comprises a set of introgression lines, each of which has asingle, possibly homozygous, marker-defined chromosomal segment thatoriginates from a donor exotic parent, in an otherwise homogenous elitegenetic background, so that the entire donor genome would be representedin a set of introgression lines. A collection of such introgressionlines is referred as libraries of introgression lines or IL libraries(ILLs). The lines of an ILL cover usually the complete genome of thedonor, or the part of interest. Introgression lines allow the study ofquantitative trait loci, but also the creation of new varieties byintroducing exotic traits. High resolution mapping of QTL using ILLsenable breeders to assess whether the effect on the phenotype is due toa single QTL or to several tightly linked QTL affecting the same trait.In addition, sub-ILs can be developed to discover molecular markerswhich are more tightly linked to the QTL of interest, which can be usedfor marker-assisted breeding (MAB). Multiple introgression lines can bedeveloped when the introgression of a single QTL is not sufficient toresult in a substantial improvement in agriculturally important traits(Gur and Zamir, Unused natural variation can lift yield barriers inplant breeding, 2004, PLoS Biol. ;2(10):e245).

Tissue Culture

As it is well known in the art, tissue culture of lettuce can be usedfor the in vitro regeneration of lettuce plants. Tissues cultures ofvarious tissues of lettuce and regeneration of plants therefrom are wellknown and published. For example, reference may be had to Teng et al.,HortScience, 27: 9, 1030-1032 (1992), Teng et al., HortScience. 28: 6,669-671 (1993), Zhang et al., Journal of Genetics and Breeding, 46: 3,287-290 (1992), Webb et al., Plant Cell Tissue and Organ Culture, 38: 1,77-79 (1994), Curtis et al., Journal of Experimental Botany, 45: 279,1441-1449 (1994), Nagata et al., Journal for the American Society forHorticultural Science, 125: 6, 669-672 (2000). It is clear from theliterature that the state of the art is such that these methods ofobtaining plants are routinely used and have a very high rate ofsuccess. Thus, another aspect of this disclosure is to provide cellswhich upon growth and differentiation produce lettuce plants having allthe physiological and morphological characteristics of lettuce cultivarCARRIZO.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollens, flowers, seeds, leaves,stems, roots, root tips, anthers, pistils, meristematic cells, axillarybuds, ovaries, seed coats, endosperms, hypocotyls, cotyledons and thelike. Means for preparing and maintaining plant tissue culture are wellknown in the art. By way of example, a tissue culture comprising organshas been used to produce regenerated plants. U.S. Pat. Nos. 5,959,185,5,973,234, and 5,977,445 describe certain techniques, the disclosures ofwhich are incorporated herein by reference.

EXAMPLES

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

Example 1- Development of Lettuce Cultivar CARRIZO Breeding History ofRomaine Lettuce CARRIZO

Lettuce cultivar CARRIZO: CARRIZO is a medium, green romaine lettucewith strong bolting tolerance and resistances to Bremia lactucae Races(BI: 7-9 US) causing downy mildew in lettuce, tomato bushy stunt virus,and lettuce aphid Nasonovia ribisnigri Biotype 0 (Nr:0). It is suitablefor production in late fall to spring seasons in the Southwest Desertsin Arizona and spring to fall seasons in the Salinas valley ofCalifornia, USA. CARRIZO has superior characteristics and was developedfrom a cross between romaine varieties.

The first cross was made in the summer of the first year of developmentof the variety between two parental varieties to obtain F1 generation ina greenhouse at Vilmorin North America (NA) Spence research facility inSalinas, CA.

The F1 seeds were grown in a greenhouse at Vilmorin NA Spence researchfacility in Salinas, CA in the fall and winter of the first year. ThreeF1 plants were identified and allowed to self and F2 seeds werecollected from each of the three F1 plants separately.

In the spring of the second year, the F2 seeds were planted in a lettucebreeding trial in Paicines, CA. Six F2 individual plants were selectedand all selected plants were allowed to self, and F3 seeds collectedindividually from each F2 plant.

In the fall of the second year, three F3 family lines were planted Yuma,AZ and a total of six individual plants were selected. All six plantswere allowed to self and seeds were collected individually from eachplant to obtain F4 seeds.

In the spring of the third year, F4 family lines were planted in abreeding trial in San Juan Bautista, CA. fourteen individual plants wereselected from two F4 family lines, and seeds were collected individuallyfrom each of the fourteen selected plants to obtain F5 seeds.

In the spring of the fourth year, F5 line was planted in San Joaquin,CA. All plants were harvested bulk and the seeds (F6) became CARRIZO.

Lettuce variety CARRIZO has been self-pollinated a number of generationswith careful evaluation and selection for uniformity of plant type andresistances to bremia, Nr0 and Tvr1, and has been increased withcontinued observation for uniformity. No variants have been observed orare expected for agronomical important traits in lettuce cultivarCARRIZO.

Lettuce cultivar CARRIZO has the following morphologic and othercharacteristics, (based primarily on data collected at the Vilmorin’sresearch station in Salinas, CA all experiments done under the directsupervision of the applicant).

TABLE 1 VARIETY DESCRIPTION INFORMATION Plant Type: Green romaine SeedColor: Black Cotyledon to Fourth Leaf Stage: Shape of cotyledon: BroadShape of fourth leaf: Elongated Apical Margin: Entire Basal Margin:Entire Undulation: Slight Green Color: Medium Green AnthocyaninDistribution: Absent Anthocyanin Concentration: N/A Rolling: PresentCupping: Uncupped Reflexing: None Harvest-Mature Out Leaf, Head, Core:Margin Incision Depth: Moderate Margin Indentation: Shallowly DentateUndulation of the Apical Margin: Moderate Green Color: Medium GreenAnthocyanin Distribution: Absent Anthoicyanin Concentration: N/AAnthocyanin Size: N/A Glossiness: Moderate Blistering: Moderate LeafThickness: Thick Trichomes: Absent (Smooth) Head Shape: Narrow ellipticHead Size Glass: Large Head Per Carton: N/A Head Firmness: Loose ButtMedrib: Flattened Maturity (No. of Days of First Water date to Harvest):Summer to Fall 65 to 105 Frame Leaf Length (cm) 25.2 cm Frame Leaf Width(cm) 17.6 cm Leaf Indez: 1.4 Leaf Area (cm²) 446.7 cm² Plant Weight(g):’ 736–1 g Plant Height (cm) 31.1 cm Core Length (cm) 4.7 cm CoreDiameter (cm) 4.2 cm Adaptation: Primary Regions of Adaptation (testedand proven adapted): Yuma, Az Season: Fall to Spring Soil Type Adaptedto most soil types, open field Diseases: Tomato bushy stynt virus:Highly resistant Downy Mildew: Highly resistant Sclerotinia Rot: Nottested Nasonovia ribisnigri: Highly resistant Physiological/Stress:Bolting: Strong bolting tolerance Tipburn: Strong tolerant

Example 2- Field Trials Characteristics of Lettuce Cultivar CARRIZO

Several traits and characteristics of lettuce cultivar CARRIZO werecompared to Abilene and Vaquero varieties. The data was collected fromvarious field locations in the United States. The field tests areexperimental trials and have been made under supervision of theapplicant. Compared to Abilene and Vaquero varieties, CARRIZO showsdifferent plant weight, plant height, frame leaf length at harvestmaturity, leaf width at harvest maturity, leaf index at harvestmaturity, leaf area at harvest maturity, core length at harvest maturityand core diameter at harvest maturity.

TABLE 2 Plant Weight (g) at Harvest Maturity Trial NO. Trial 1 Trial 2Trial 3 Abilene 503 911 646 410 844 673 508 835 623 336 802 503 568 824669 392 780 719 536 687 629 411 803 769 490 728 668 524 792 699 Vaquero624 701 716 553 701 803 781 800 769 738 662 905 539 785 954 834 711 654724 591 681 646 679 1055 550 702 788 789 800 900 Carrizo 868 742 490 980641 420 925 523 623 679 1006 603 949 874 743 716 437 801 724 697 819 932720 440 1113 889 710 936 650 433 Anova: Two-Factor With Replication

SUMMARY Abilene: Abilene Trial 1 Trial 2 Trial 3 Total Count 10 10 10 30Sum 4678 8006 6598 19282 Average 467.8 800.6 659.8 642.733333 Variance5653.5111 3831.6 4930.17778 23720.2023 Vaquero: Vaquero Count 10 10 1030 Sum 6778 7132 8225 22135 Average 677.8 713.2 822.5 737.833333Variance 12027.956 4375.067 16483.3889 14130.0057 Carrizo: ROM 18526Count 10 10 10 30 Sum 8822 7179 6082 22083 Average 882.2 717.9 608.2736.1 Variance 18738.178 29453.43 24307.2889 35615.1276 Total Count 3030 30 Sum 20278 22317 20905 Average 675.93333 743.9 696.833333 Variance40912.616 13354.3 22816.6264

ANOVA Source of Variation SS df MS F P-value F critical value Variety177643.49 2 88821.7444 6.67271867 0.002077 3.109310547 Location72715.489 2 36357.7444 2.73136946 0.0711327 3.109310547 Interaction979573.84 4 244893.461 18.3975802 8.686E-11 2.48444144 Within 1078205.481 13311.1778 Total 2308138.2 89 Anova shows significant differences inplant weight in varieties and interactions between variety and location,and no significant difference in locations. The average plant weight (g)of Abilene, Vaquero and Carrizo are 642.7, 737.8 and 736.1,respectively.

TABLE 3 Plant Height (cm) at Harvest Maturity Trial NO. Trial 1 Trial 2Trial 3 Abilene 31 35 34 26 36 36 30 36 35 26 36 33 30 37 33 29 38 33 3035 32 27 35.5 33 31 36 33 29 37 35 Vaquero 30 34 27 27 33.5 30 26 34 3025 34 32 28 36 32 31 34 31 30 32 30 26 33 30 25 34 30 27 32 32 Carrizo29 33 31 29 32 30 32 33 29 30 34 30 32 33 29 29 33 30 30 34 31 30 33 3031 34 29 30 33 30 Anova: Two-Factor With Replication

SUMMARY Abilene Trial 1 Trial 2 Trial 3 Total Abilene Count 10 10 10 30Sum 289 361.5 337 987.5 Average 28.9 36.15 33.7 32.9166667 Variance3.6555556 0.891667 1.56666667 11.2772989 Vaquero Count 10 10 10 30 Sum275 336.5 304 915.5 Average 27.5 33.65 30.4 30.5166667 Variance4.7222222 1.336111 2.26666667 9.11178161 Carrizo Count 10 10 10 30 Sum302 332 299 933 Average 30.2 33.2 29.9 31.1 Variance 1.2888889 0.40.54444444 2.98965517 Count 30 30 30 Sum 866 1030 940 Average 28.86666734.33333 31.3333333 Variance 4.2574713 2.557471 4.29885057

ANOVA Source of Variation SS df MS F P-value F critical value Variety94.005556 2 47.0027778 25.3730423 2.781E-09 3.109310547 Location449.68889 2 224.844444 121.375541 4.267E-25 3.109310547 Interaction78.244444 4 19.5611111 10.5594802 6.182E-07 2.48444144 Within 150.05 811.85246914 Total 771.98889 89 Anova shows significant differences inplant height for varieties, locations and interactions between varietyand location. The average plant height (cm) of Abilene, Vaquero andCarrizo are 32.9, 30.5 and 31.1, respectively.

TABLE 4 Frame Leaf Length (cm) at Harvest Maturity Trial NO. Trial 1Trial 2 Trial 3 Abilene 24 29 30 26 25 29 25 29 28 22 28 25 24 29 27 2326 26 24 27 23 26 29 20 22 30 24 25 26 22 Vaquero 23 24 29 23 24 21 2427 29 25 24 25 26 27 31 25 27 26 22 26 28 21 28 26 25 27 29 25 24 27Carrizo 22 26 22 21 29 28 25 29 27 28 24 25 27 20 25 25 25 27 23 26 2821 25 26 29 26 25 22 28 21 Anova: Two-Factor With Replication

SUMMARY Trial 1 Trial 2 Trial 3 Total Abilene Count 10 10 10 30 Sum 241278 254 773 Average 24.1 27.8 25.4 25.7666667 Variance 2.1 2.84444410.2666667 7.15057471 Vaquero Count 10 10 10 30 Sum 239 258 271 768Average 23.9 25.8 27.1 25.6 Variance 2.5444444 2.622222 7.877777785.83448276 Carrizo Count 10 10 10 30 Sum 243 258 254 755 Average 24.325.8 25.4 25.1666667 Variance 8.6777778 7.066667 5.6 7.04022989 TotalCount 30 30 30 Sum 723 794 779 Average 24.1 26.46667 25.9666667 Variance4.162069 4.809195 8.03333333

ANOVA Source of Variation SS df MS F P-value F critical value Variety5.7555556 2 2.87777778 0.52217742 0.5952107 3.109310547 Location93.355556 2 46.6777778 8.46975806 0.0004569 3.109310547 Interaction40.977778 4 10.2444444 1.85887097 0.1256694 2.48444144 Within 446.4 815.51111111 Total 586.48889 89 Anova shows no significant differences inframe leaf length at harvest maturity stage for varieties andinteractions between variety and location, but significant differencesfor locations. The average matured leaf length (cm) of Abilene, Vaqueroand Carrizo are 25.8, 25.6 and 25.2, respectively.

TABLE 5 Leaf Width (cm) at Harvest Maturity Trial NO. Trial 1 Trial 2Trial 3 Abilene 13 17 18 16 15 22 13.5 20 19 11 19 20 12 21 20 13.5 1819 13.5 19 19 15 18 18 14 18 17 14 18 17 Vaquero 13 15 18 18 14 19 14 1719 18 15 18 14 16 21 17 15 18 16 14 18 16 15 21 16 15 20 15 14 21Carrizo 16 17 16 14 18 20 16 22 17 19 18 18 14 17 19 17 18 18 13 19 1913 19 20 19 18 23 15 17 20 Anova: Two-Factor With Replication

SUMMARY Trial 1 Trial 2 Trial 3 Total Abilene Count 10 10 10 30 Sum135.5 183 189 507.5 Average 13.55 18.3 18.9 16.9166667 Variance1.9694444 2.677778 2.32222222 8.08764368 Vaquero Count 10 10 10 30 Sum157 150 193 500 Average 15.7 15 19.3 16.6666667 Variance 2.9 0.8888891.78888889 5.40229885 Carrizo Count 10 10 10 30 Sum 156 183 190 529Average 15.6 18.3 19 17.6333333 Variance 4.9333333 2.233333 3.777777785.61954023 Total Count 30 30 30 Sum 448.5 516 572 Average 14.95 17.219.0666667 Variance 4.0577586 4.303448 2.47816092

ANOVA Source of Variation SS df MS F P-value F critical value Variety15.105556 2 7.55277778 2.89357928 0.0611219 3.109310547 Location254.93889 2 127.469444 48.8354026 1.218E-14 3.109310547 Interaction87.811111 4 21.9527778 8.41042923 1.001E-05 2.48444144 Within 211.425 812.61018519 Total 569.28056 89 Anova shows no significant differences inframe leaf width at harvest maturity stage for varieties, but showssignificant differences for locations and interactions between varietyand location. The average matured leaf width (cm) of Abilene, Vaqueroand Carrizo are 16.9, 16.7 and 17.6, respectively.

TABLE 6 Leaf Index calculated by dividing the leaf length by the leafwidth Trial NO. Trial 1 Trial2 Trial3 Abilene 1.85 1.71 1.67 1.63 1.671.32 1.85 1.45 1.47 2.00 1.47 1.25 2.00 1.38 1.35 1.70 1.44 1.37 1.781.42 1.21 1.73 1.61 1.11 1.57 1.67 1.41 1.79 1.44 1.29 Vaquero 1.77 1.601.61 1.28 1.71 1.11 1.71 1.59 1.53 1.39 1.60 1.39 1.86 1.69 1.48 1.471.80 1.44 1.38 1.86 1.56 1.31 1.87 1.24 1.56 1.80 1.45 1.67 1.71 1.29Carrizo 1.38 1.53 1.38 1.50 1.61 1.40 1.56 1.32 1.59 1.47 1.33 1.39 1.931.18 1.32 1.47 1.39 1.50 1.77 1.37 1.47 1.62 1.32 1.30 1.53 1.44 1.091.47 1.65 1.05 Anova: Two-Factor With Replication

SUMMARY Trial 1 Trial2 Trial3 Total Abilene Count 10 10 10 30 Sum17.894963 15.2649 13.4544735 46.6143418 Average 1.7894963 1.526491.34544735 1.55381139 Variance 0.0201112 0.014782 0.02335317 0.05245914Vaquero Count 10 10 10 30 Sum 15.394581 17.22812 14.0815789 46.7042761Average 1.5394581 1.722812 1.40815789 1.5568092 Variance 0.0417160.011147 0.0245115 0.04123869 Carrizo Count 10 10 10 30 Sum 15.68794214.13311 13.4785544 43.2996074 Average 1.5687942 1.413311 1.347855441.44332025 Variance 0.0272265 0.021319 0.02911194 0.03298264 Total Count30 30 30 Sum 48.977486 46.62613 41.0146069 Average 1.6325829 1.5542041.36715356 Variance 0.0405212 0.031576 0.02475996

ANOVA Source of Variation SS df MS F P-value F critical value Variety0.2509702 2 0.12548512 5.29525228 0.0068976 3.109310547 Location1.1158392 2 0.55791959 23.54323 8.705E-09 3.109310547 Interaction0.6383834 4 0.15959584 6.73466521 9.899E-05 2.48444144 Within 1.919510981 0.02369767 Total 3.9247037 89 Anova shows significant differences inframe leaf index at harvest maturity stage for varieties, locations andinteractions between variety and location. The average matured leafindex of Abilene, Vaquero and Carrizo are 1.5, 1.5 and 1.4,respectively.

TABLE 7 Leaf Area (cm2), calculated by multiplying the leaf length bythe leaf width Trial NO. Trial 1 Trial2 Trial3 Abilene 312 493 540 416375 638 337.5 580 532 242 532 500 288 609 540 310.5 468 494 324 513 437390 522 360 308 540 408 350 468 374 Vaquero 299 360 522 414 336 399 336459 551 450 360 450 364 432 651 425 405 468 352 364 504 336 420 546 400405 580 375 336 567 Carrizo 352 442 352 294 522 560 400 638 459 532 432450 378 340 475 425 450 486 299 494 532 273 475 520 551 468 575 330 476420 Anova: Two-Factor With Replication

SUMMARY Trial 1 Trial2 Trial3 Total Trial No. Count 10 10 10 30 Sum 32785100 4823 13201 Average 327.8 510 482.3 440.033333 Variance 2459.56674253.333 7564.45556 11078.5333 Vaquero Count 10 10 10 30 Sum 3751 38775238 12866 Average 375.1 387.7 523.8 428.866667 Variance 2193.21111792.233 5218.62222 7545.36092 Carrizo Count 10 10 10 30 Sum 3834 47374829 13400 Average 383.4 473.7 482.9 446.666667 Variance 9260.93335662.233 4558.98889 8131.12644 Total Count 30 30 30 Sum 10863 1371414890 Average 362.1 457.1333 496.333333 Variance 4938.4552 6354.2575772.29885

ANOVA Source of Variation SS df MS F P-value F critical value Variety4855.3556 2 2427.67778 0.50854936 0.6032745 3.109310547 Location285865.62 2 142932.811 29.9415311 1.84E-10 3.109310547 Interaction103357.78 4 25839.4444 5.41284064 0.0006502 2.48444144 Within 386672.281 4773.73086 Total 780750.96 89 Anova shows significant differences inleaf area (cm²) at harvest maturity stage for locations and interactionsbetween variety and location, and shows no significant difference forvarieties. The average leaf area (cm²) of Abilene, Vaquero and Carrizoare 440.0, 428.9 and 446.7, respectively.

TABLE 8 Core Length (cm) at Harvest Maturity Trial NO. Trial 1 Trial 2Trial 3 Abilene 4.5 6.0 5.0 5.0 5.0 4.0 4.0 6.0 4.0 5.0 4.0 4.0 4.0 4.54.0 5.0 5.0 5.0 5.0 4.0 5.0 5.5 8.0 5.0 4.0 5.0 5.0 4.0 4.0 5.0 Vaquero4.0 5.0 6.0 5.5 4.0 4.0 5.0 5.0 5.0 5.0 6.0 5.5 4.5 4.0 4.0 4.0 4.0 4.04.5 5.0 3.0 6.0 5.0 4.0 3.5 5.0 3.0 3.5 4.0 5.0 Carrizo 5.0 6.5 3.0 5.05.0 4.0 6.0 4.5 5.0 4.0 3.0 5.5 7.0 4.0 5.0 4.0 4.0 5.0 6.0 4.0 3.0 8.04.5 3.0 5.0 4.0 3.5 7.0 4.5 4.0 Anova: Two-Factor With Replication

SUMMARY Trial 1 Trial 2 Trial 3 Total Abilene Count 10 10 10 30 Sum 4651.5 46 143.5 Average 4.6 5.15 4.6 4.78333333 Variance 0.32222221.558333 0.26666667 0.73591954 Vaquero Count 10 10 10 30 Sum 45.5 4743.5 136 Average 4.55 4.7 4.35 4.53333333 Variance 0.6916667 0.4555561.00277778 0.68850575 Carrizo Count 10 10 10 30 Sum 57 44 41 142 Average5.7 4.4 4.1 4.73333333 Variance 1.7888889 0.822222 0.93333333 1.59885057Total Count 30 30 30 Sum 148.5 142.5 130.5 Average 4.95 4.75 4.35Variance 1.1612069 0.978448 0.72672414

ANOVA Source of Variation SS df MS F P-value F critical value Variety1.05 2 0.525 0.60255048 0.5498487 3.109310547 Location 5.6 2 2.83.21360255 0.0453896 3.109310547 Interaction 11.5 4 2.875 3.299681190.0147656 2.48444144 Within 70.575 81 0.8712963 Total 88.725 89 Anovashows significant differences in core length at harvest maturity stagefor locations and interactions between variety and location, and showsno significant difference for varieties. The average core length (cm) ofAbilene, Vaquero and Carrizo are 4.8, 4.5 and 4.7, respectively.

TABLE 9 Core Diameter (cm) at Harvest Maturity Trial NO. Trial 1 Trial 2Trial 3 Abilene 3.0 4.0 4.5 4.0 3.5 4.0 3.5 4.0 4.0 4.0 4.0 4.5 4.0 4.03.5 4.0 4.5 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 5.0 4.0 3.5 5.0 3.5 Vaquero3.0 4.0 4.5 4.0 4.0 4.0 4.0 4.0 4.0 3.5 5.0 5.5 4.0 3.0 4.0 4.0 4.0 4.04.0 4.0 3.5 3.0 4.0 4.0 3.5 4.0 6.0 3.5 4.0 4.0 Carrizo 4.0 4.0 3.0 4.04.0 4.0 5.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 5.0 4.0 5.0 4.5 5.0 3.0 6.04.5 3.0 5.0 4.5 3.0 4.0 4.0 4.0 Anova: Two-Factor With Replication

SUMMARY Trial 1 Trial 2 Trial 3 Total Abilene Count 10 10 10 30 Sum 3842 40 120 Average 3.8 4.2 4 4 Variance 0.1222222 0.233333 0.111111110.17241379 Vaquero Count 10 10 10 30 Sum 36.5 40 43.5 120 Average 3.65 44.35 4 Variance 0.1694444 0.222222 0.61388889 0.39655172 Carrizo Count10 10 10 30 Sum 45.5 42 37 124.5 Average 4.55 4.2 3.7 4.15 Variance0.4694444 0.122222 0.45555556 0.45086207 Total Count 30 30 30 Sum 120124 120.5 Average 4 4.133333 4.01666667 Variance 0.3965517 0.1885060.43936782

ANOVA Source of Variation SS df MS F P-value F critical value Variety0.45 2 0.225 0.80374862 0.4511853 3.109310547 Location 0.3166667 20.15833333 0.56560088 0.5702458 3.109310547 Interaction 6.5833333 41.64583333 5.87927233 0.0003322 2.48444144 Within 22.675 81 0.27993827Total 30.025 89 Anova shows no significant differences in core diameterat harvest maturity stage for varieties and locations, and showssignificant interactions between variety and location. The average corediameter (cm) of Abilene, Vaquero and Carrizo are 4.0, 4.0 and 4.2,respectively.

DEPOSIT INFORMATION

A deposit of the lettuce seed of this disclosure is maintained byVilmorin-Mikado USA, Inc., 3 Harris Place, Salinas, California 93901,USA. In addition, a sample of the lettuce seed of this disclosure hasbeen deposited with the National Collections of Industrial, Food andMarine Bacteria (NCIMB), NCIMB Ltd. Ferguson Building, CraibstoneEstate, Bucksburn, Aberdeen, AB21 9YA Scotland, United Kingdom.

To satisfy the enablement requirements of 35 U.S.C. 112, and to certifythat the deposit of the isolated strain of the present disclosure meetsthe criteria set forth in 37 C.F.R. 1.801-1.809, Applicants hereby makethe following statements regarding the deposited lettuce cultivarCARRIZO (deposited as NCIMB Accession No.______):

-   1. During the pendency of this application, access to the disclosure    will be afforded to the Commissioner upon request;-   2. All restrictions on availability to the public will be    irrevocably removed upon granting of the patent under conditions    specified in 37 CFR 1.808;-   3. The deposit will be maintained in a public repository for a    period of 30 years or 5 years after the last request or for the    effective life of the patent, whichever is longer;-   4. A test of the viability of the biological material at the time of    deposit will be conducted by the public depository under 37 C.F.R.    1.807; and-   5. The deposit will be replaced if it should ever become    unavailable.

Access to this deposit will be available during the pendency of thisapplication to persons determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. § 1.14 and 35 U.S.C. §122. Upon allowance of any claims in this application, all restrictionson the availability to the public of the variety will be irrevocablyremoved by affording access to a deposit of at least 625 seeds of thesame variety with the NCIMB.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes.

However, mention of any reference, article, publication, patent, patentpublication, and patent application cited herein is not, and should notbe taken as an acknowledgment or any form of suggestion that theyconstitute valid prior art or form part of the common general knowledgein any country in the world.

What is claimed is:
 1. A seed of lettuce designated CARRIZO, wherein arepresentative sample of seed of said lettuce has been deposited underNCIMB No.______.
 2. A lettuce plant, a plant part thereof, or a plantcell thereof, produced by growing the seed of claim 1, wherein thelettuce plant or a lettuce plant regenerated from said plant part orplant cell has all the physiological and morphological characteristicsof lettuce designated CARRIZO deposited under NCIMB No.______.
 3. Thelettuce plant, the plant part thereof, or the plant cell thereof ofclaim 2, wherein the plant part is selected from the group consisting ofa leaf, a flower, a head, a seed, and a cell.
 4. A lettuce plant, aplant part thereof, or a plant cell thereof, wherein the lettuce plantor a plant regenerated from the plant part or the plant cell has all thephysiological and morphological characteristics of lettuce designatedCARRIZO deposited under NCIMB No.______.
 5. A tissue culture ofregenerable cells produced from the lettuce plant or the plant partthereof of claim 2, wherein a plant regenerated from the tissue culturehas all the physiological and morphological characteristics of lettucedesignated CARRIZO deposited under NCIMB No.______.
 6. A lettuce plantregenerated from the tissue culture of claim 5, said plant having allthe physiological and morphological characteristics of lettucedesignated CARRIZO deposited under NCIMB No.______.
 7. A lettuce head orleaf produced from the plant of claim
 2. 8. A method for harvesting alettuce head, said method comprising (a) growing the lettuce plant ofclaim 2 to produce a lettuce head, and (b) harvesting said lettuce head.9. A lettuce head produced by the method of claim
 8. 10. A method forharvesting a lettuce leaf, said method comprising: (a) growing thelettuce plant of claim 2 to produce a lettuce leaf, and (b) harvestingsaid lettuce leaf.
 11. A lettuce leaf produced by the method of claim10.
 12. A method for producing a lettuce seed, said method comprising(a) crossing a first parent lettuce plant with a second parent lettuceplant and (b) harvesting the resultant lettuce seed, wherein said firstparent lettuce plant and/or second parent lettuce plant is the lettuceplant of claim
 2. 13. An F1 lettuce seed produced by the method of claim12.
 14. A method for producing a lettuce seed, said method comprising(a) self-pollinating the lettuce plant of claim 2 and (b) harvesting theresultant lettuce seed.
 15. A lettuce seed produced by the method ofclaim
 14. 16. A method of producing a lettuce plant derived from thelettuce designated CARRIZO, said method comprising: (a) crossing theplant of claim 2 with a second lettuce plant to produce a progeny plant.17. The method of claim 16 further comprising the steps of: (b) crossingthe progeny plant derived from lettuce designated CARRIZO with itself ora second lettuce plant to produce a seed of progeny plant of subsequentgeneration; (c) growing the progeny plant of the subsequent generationfrom the seed; (d) crossing the progeny plant of the subsequentgeneration with itself or a second lettuce plant to produce a lettuceplant derived from the lettuce designated CARRIZO and (e) repeating step(c) and/or (d) for at least one generation to produce a lettuce plantderived from the lettuce designated CARRIZO.
 18. A method of producing aplant of lettuce designated CARRIZO comprising at least one desiredtrait, the method comprising introducing a single locus conversionconferring the desired trait into lettuce designated CARRIZO, whereby aplant of lettuce designated CARRIZO comprising the desired trait isproduced.
 19. A lettuce plant, a part thereof, or a cell thereof,produced by the method of claim 18, wherein the plant, the part thereof,or the cell thereof comprises a single locus conversion and essentiallyall of the physiological and morphological characteristics of CARRIZOdeposited under NCIMB No.______.
 20. The plant of claim 19, wherein thesingle locus conversion confers said plant with herbicide resistance,insect resistance, resistance for bacterial, fungal, mycoplasma or viraldisease, drought or salt tolerance, water-stress tolerance, heat-stresstolerance, enhanced plant vigor, improved shelf life, delayed senescenceor controlled ripening, enhanced nutritional quality, increased texture,improved yield and recovery and/or improved fresh cut application. 21.The plant of claim 19, wherein the single locus conversion is introducedinto the plant by the use of recurrent selection, mutation breeding,wherein said mutation breeding selects for a mutation that isspontaneous or artificially induced, backcrossing, pedigree breeding,haploid/double haploid production, marker-assisted selection, genetictransformation, genomic selection, synthetic genomics, Zinc fingernuclease (ZFN), oligonucleotide directed mutagenesis, cisgenesis,intragenesis, RNA-dependent DNA methylation, agro-infiltration,Transcription Activation-Like Effector Nuclease (TALEN), CRISPR/Cassystem, engineered meganuclease, engineered homing endonuclease, and/orDNA guided genome editing.
 22. A method for producing nucleic acids,said method comprising isolating nucleic acids from the plant of claim2, or a plant part, or a plant cell thereof.
 23. A method for producinga second lettuce plant, said method comprising applying plant breedingtechniques to the plant or plant part of claim 2 to produce the secondlettuce plant.
 24. A method of introducing a desired trait into lettuceCARRIZO, said method comprising the steps of: (a) crossing a lettucedesignated CARRIZO plant grown from a seed of lettuce designated CARRIZOdeposited under NCIMB No.______, with another lettuce plant thatcomprises a desired trait to produce F1 progeny plants; (b) selectingone or more progeny plants that have the desired trait to produceselected progeny plants; (c) crossing the selected progeny plants withthe lettuce designated CARRIZO plants to produce backcross progenyplants; (d) selecting for the backcross progeny plants that have thedesired trait and essentially all the physiological and morphologicalcharacteristics of lettuce designated CARRIZO deposited under NCIMBNo.______ to produce selected backcross progeny plants; and (e)repeating steps (c) and (d) at least one time in succession to produceselected backcross progeny plants that comprise the desired trait andessentially all of the physiological and morphological characteristicsof lettuce designated CARRIZO deposited under NCIMB No.______.